JP2022543367A - joining member - Google Patents

Abstract

The present invention relates to specific binding members, such as antibodies and fragments thereof, capable of specifically binding Lewis Y (Ley) carbohydrates. The invention also relates to the use of said binding members in medicine and nucleic acids encoding said binding members. [Selection figure] None

Description

The present invention relates to specific binding members, such as antibodies and fragments thereof, capable of specifically binding Lewis Y (Le ^y ) carbohydrates. The invention also relates to the use of said binding members in medicine and nucleic acids encoding said binding members.

Glycan structures are present in both protein and glycolipid backbones and can be significantly overexpressed in cancer due to altered expression of glycosyltransferases. Glycolipids consist of a lipid tail with a carbohydrate head and make up 5% of the lipid molecules in the outer monolayer. There are three major glycolipid series, the globo, neolacto, and ganglio lipids. Glycolipids are postulated to be very good targets due to their high surface density, mobility, and association with membrane microdomains, all of which contribute to strong cellular interactions. However, the generation of anti-glycolipid antibodies is a difficult task as there is no T-cell assistance and monoclonal antibodies (mAbs) are usually of low affinity and IgM subclass (Durrant, Noble et al. 2012). , Rabu, McIntosh et al. 2012). Although mAbs directed against protein-expressed glycans overcome this problem, mAbs rarely see only small glycans and usually recognize specific protein glycans, providing very restricted expression. As such, they present new challenges.

Only a limited number of antibodies that recognize glycans have been described. Several anti-Lewis (Le) carbohydrate antigen mAbs have been generated to date, but these often have cross-reactivity with a range of glycans expressed in normal tissues. Lewis carbohydrate antigens are formed by the sequential addition of fucose onto chains of oligosaccharide precursors on glycoproteins or glycolipids through the action of a set of glycosyltransferases (Yuriev, Farrugia et al. 2005). They can be divided into two groups, type I (Le ^a and Le ^b ) and type II (Le ^x and Le ^y ). The Le ^a and Le ^b antigens have been identified as blood group antigens, and Le ^x and Le ^y have been reported to be expressed only in specific epithelial cells (Scott, Geleick et al. 2000) and have been associated with tumors. It is considered a relevant marker (Soejima and Koda 2005) and represents an attractive target for cancer treatments, including antibody-based immunotherapy (Scott, Geleick et al. 2000). Most normal tissues do not express Lewis Y, except epithelial cells of the gastrointestinal tract, which, however, are of very low density (Schuster, Umana et al. 2005). However, Lewis Y (Fucal-2Galb1-4(Fucal-3)GlcNAc) has been reported to be highly densely expressed in nearly 70% of human epithelial carcinomas, thus making it attractive for cancer immunotherapy. candidate target. Recognition of Lewis ^Y as a good therapeutic target has led to the production of many anti-Ley antibodies. Some of these anti-Le ^y mAbs cross-react with other Lewis antigens, such as mAb 692/29, which recognizes Le ^b/y , and have shown anti-tumor responses against colon tumors in xenograft models ( Noble, Spendlove et al. 2013).

Several other ^mAbs that bind Ley are known in the art, but these cross-react with other related Lewis antigens. For example, European Patent Publication No. 0285059 discloses an antibody, BR-55, which reacts with both Lewis ^y and B-7-2. B-7-2 has also been shown to be associated with tumor cells (European Patent Publication No. 0285059). US Pat. No. 5,869,045 discloses an antibody, BR-96, that binds to both the Lewis ^y and Lewis ^x haptens. Antibodies that bind to both Lewis ^y and Lewis ^b antigens are known. Studies have shown that the C14 monoclonal antibody recognizes and binds to both Lewis ^y and Lewis ^b (extended and unextended forms) antigens (Durrant at al., Hybridoma, 12 , 647-660 (1996)). WO 02/092126 discloses mAbs that bind to both the Lewis ^y and Lewis ^b haptens.

A number of clinical trials have been conducted utilizing murine or chimeric anti-Ley antibodies (Scott, ^Geleick et al. 2000). Unfortunately, anti-Ley mAbs often cross-react with Lewis X and type H II structures that are more commonly expressed in normal tissues (Furukawa, Welt et al. 1990, Yin, ^Finstad et al. .. 1996). A range of Le ^y antibodies have also been identified, but a consistent problem with these antibodies is their degree of cross-reactivity with Le ^x and H2-type structures, which can lead to erythrocyte agglutination and gastrointestinal toxicity. (Kitamura, Stockert et al. 1994, Pai, Wittes et al. 1996, Tolcher, Sugarman et al. 1999). Thus, fine specificity of anti-Le ^y antibodies is critical to successful clinical treatment.

In a first aspect, the invention provides an isolated specific binding member capable of specifically binding Fucal-2Galb1-4(Fucal-3)GlcNAc(Le ^y ).

The inventors have provided specific binding members that exhibit potent anti-tumor activity in vivo. The specific binding members of the present invention exhibit potent immune-mediated cytotoxic activity against human colon cancer cells in vitro via antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). present. They also have the property of directly inducing cell death without the need for immune effector cells. By immunizing with glycolipids rather than tumor glycoproteins or whole cells, we generated hyperspecific anti-Le ^y mAbs that did not cross-react with any other glycans. Examples of these mAbs include the IgG3k mouse mAb known herein as "FG27.10" and the IgG1k mouse mAb known herein as "FG27.18", the chimeric hIgG1 (as "CH27" known as "Hu27") and humanized hIgG1 (known as "Hu27").

In contrast to previous studies with anti-Le ^y mAbs that cross-reacted with other glycans, the specific binding members of the present invention had a very restricted normal distribution and were found in normal stomach, lung, Binds only to a subpopulation of cells in the tonsils, pancreas, and duodenum. Furthermore, they were unable to stain colon, jejunum, breast, kidney, or ileum. They bind potently to a wide range of tumors, including 80% gastric, 100% colorectal, 95% ovarian, and 85% breast tumors. A specific binding member of the invention, eg FG27.10/18, has monospecificity only for Le ^y . The inventors have shown that specific binding members according to the invention were unable to bind to the cell line Colo21. In contrast, the Le ^y/x mAb bound strongly and the Le ^y/b mAb moderately. This indicates that Colo201 expresses Le ^x and Le ^b , but not Le ^y , confirming the specificity of the specific binding members of the invention. This specificity is reflected in similar but separate sequences of the FG27 variable heavy and light chain regions (Fig. 13a,b). FG27 differs from other cross-reactive Le ^y mabs by 16-68% differences in amino acid sequence CDRs in the heavy chain and 4.3-16% differences in amino acid differences in CDR sequences in the light chain. , 8.6-49.5% amino acid sequence differences in the variable region of the heavy chain, and 2-24% difference in the amino acid sequence of the variable region of the light chain (see Table 4 herein). Most of the variation is in CDRH2, with 3-5 of the 8 amino acids varying between different mAbs.

Specific binding members of the invention may bind to lipid or protein scaffolds. The protein backbone can have a molecular weight of approximately 50-150 kDa, as determined by SDS-PAGE.

A specific binding member of the invention preferably has the amino acid sequence substantially represented as residues 27-38 (CDRH1), 54-65 (CDRH2), or 105-116 (CDRH3) of FIG. 1a or 1b. comprising one or more binding domains selected from binding domains having A specific binding member may comprise a binding domain comprising an amino acid sequence substantially represented as residues 105-116 (CDRH3) of the amino acid sequence of Figures 1a or 1b. Such specific binding members are binding domains having amino acid sequences substantially represented as residues 27-38 (CDRH1) and residues 56-65 (CDRH2) of the amino acid sequences shown in Figures 1a and 1b. may further comprise one or both, preferably both, of

A binding member may comprise an amino acid sequence substantially presented as 1-127 (VH) in Figures 1a or 1b.

The specific binding member is one or more binding domains selected from binding domains having the amino acid sequence of residues 27-38 (CDRL1), 56-65 (CDRL2), or 105-113 (CDRL3) of Figure 1c can include The binding member may comprise a binding domain having an amino acid sequence substantially represented as residues 105-113 (CDRL3) of the amino acid sequence of Figure 1c. Such specific binding members are among binding domains having amino acid sequences substantially represented as residues 27-38 (CDRL1) and residues 56-65 (CDRL2) of the amino acid sequence shown in Figure 1c. may further include one or both, preferably both.

Specific binding members comprising multiple binding domains of the same or different sequences, or combinations thereof, are included within the scope of the invention. At least one binding domain may be carried by a human antibody framework. For example, one or more of the binding domains may be replaced with the complementarity determining regions (CDRs) of a human whole antibody or variable region thereof.

One isolated specific binding member of the invention comprises an amino acid sequence substantially represented as residues 1-124 (VL) of the amino acid sequence shown in Figure 1c.

An isolated specific binding member of the invention has an amino acid sequence substantially represented as residues 27-38 (CDRL1), 56-65 (CDRL2) or 105-113 (CDRL3) of Figure 1c. substantially represented as residues 27-38 (CDRH1), 54-65 (CDRH2) or 105-116 (CDRH3) of FIG. 1a or 1b in combination with one or more, preferably all, of the binding domains It may contain one or more, preferably all, of the binding domains having the amino acid sequence.

The binding member is represented substantially as residues 1-127 (VH) of the amino acid sequence of FIG. 1a or 1b and substantially as residues 1-124 (VL) of the amino acid sequence of FIG. 1c. and an amino acid sequence that is

Having isolated a single prototypic mAb, such as the FG27 mAb, with the desirable properties described herein, other prototypic mAbs with similar properties can be isolated using methods known in the art. Making mAbs is easy. For example, Jespers et al. , 1994 (Jespers, Roberts et al. 1994) can be used to guide the selection of mAbs with the same epitopes and thus similar properties as the prototype mAb. Using phage display, the first heavy chain of the prototypic antibody is paired with a repertoire of (preferably human) light chains to select mAbs that bind the glycan, and then the novel light chain is prototypic are paired with a repertoire of (preferably human) heavy chains in order to select mAbs that bind to (preferably human) glycans with the same epitope as the mAbs of .

A specific binding member can be an antibody or antibody fragment, Fab, (Fab')2, scFv, Fv, dAb, Fd, or diabodies. Antibodies can be polyclonal antibodies. Antibodies can be monoclonal antibodies (mAbs). Antibodies of the invention may be humanized, chimeric, or veneered, or may be non-human antibodies of any species.

Murine or chimeric antibodies carry an increased risk of adverse anti-mouse antibody (HAMA) responses in patients (Schroff et al. 1985; Azinovic et al. 2006; Miotti et al. 1999; D'Arcy and Mannik 2001). Thus, most approved therapeutic mAbs are humanized or fully human IgG antibodies.

A specific binding member of the invention may comprise a heavy chain having an amino acid sequence substantially as presented in Figure 2a and a light chain having an amino acid sequence substantially as presented in Figure 2b.

A specific binding member of the invention may comprise a heavy chain having an amino acid sequence substantially as presented in Figure 3a and a light chain having an amino acid sequence substantially as presented in Figure 3b.

A specific binding member of the invention may comprise a heavy chain having an amino acid sequence substantially as presented in FIG. 33 and/or a light chain having an amino acid sequence substantially as presented in FIG.

Further, the present invention provides an antibody comprising a VH chain having the amino acid sequence of residues 1-127 of FIG. 1a or 1b and a VL chain having the amino acid sequence of residues ^1-124 of FIG. Providing binding members that compete for binding.

A specific binding member capable of specifically binding to ^Ley and which is at least 90%, at least 95%, or at least 99% identical in VH and/or VL domains to the VH or VL domains of Figures 1, 2, or 3 , are included in the present invention. Specific binding members that are capable of specifically binding Le ^y and that are at least 90%, at least 95%, or at least 99% identical to the heavy and/or light chains of Figure 33 are included in the present invention. Preferably, such antibodies differ from the sequences of Figures 1, 2, or 3 by minor functionally insignificant amino acid substitutions (eg, conservative substitutions), deletions, or insertions.

A specific binding member of the invention may carry a detectable or functional label.

In a further aspect, the invention comprises isolated nucleic acid encoding a specific binding member of the invention and expressing said nucleic acid under conditions conducive to expression of said binding member and recovering the binding member. provides methods for preparing specific binding members of the invention. An isolated nucleic acid encoding a specific binding member capable of specifically binding ^Ley and at least 90%, at least 95%, or at least 99% identical to a sequence provided herein is an isolated nucleic acid of the present invention. include.

A specific binding member of the invention is a method of treatment or diagnosis of the human or animal body, e.g. a method of treatment of a tumor in a patient (preferably human), wherein an effective amount of a specific binding member of the invention is It can be used in a method comprising administering to the patient. The invention also provides a specific binding member of the invention for use in medicine, preferably in the treatment of tumors, and a specific binding member of the invention in the manufacture of a medicament for the diagnosis or treatment of tumors. Provide member use. The tumor may be a gastric, colorectal, pancreatic, lung, ovarian, or breast tumor.

Disclosed herein are antigens to which specific binding members of the invention bind. A specific binding member of the invention may preferably provide a Le ^y capable of specifically binding. Le ^y may be provided in isolated form and used in screening to develop additional specific binding members thereto. For example, a library of compounds can be screened for members of the library that specifically bind to ^Ley . The Le ^y can be on a lipid or protein backbone. When on a protein backbone, it can have a molecular weight of about 50-150 kDa, as determined by SDS-PAGE.

In a further aspect, the present invention provides a ^Ley -containing glycan, preferably according to the first aspect of the invention, for use in the diagnosis or prognosis of gastric, colorectal, pancreatic, lung, ovarian and breast tumors. An isolated specific binding member capable of binding is provided.

The invention further provides a method of diagnosing cancer comprising using a specific binding member of the invention to detect ^Ley -containing glycans in a sample derived from an individual. In this diagnostic method, the glycan pattern detected by the binding member can be used to stratify therapeutic options for an individual.

These and other aspects of the invention are described in further detail below.

As used herein, a "specific binding member" is a member of a pair of molecules that have binding specificity for each other. The members of a specific binding pair may be of natural origin or wholly or partly synthetically produced. One member of a molecular pair may be a protrusion or cavity on its surface that binds specifically to a particular spatial and polar organization of the other member of the molecular pair and is thus complementary thereto. has an area of Members of the pair thus have the property of binding specifically to each other. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, 5 receptor-ligand, enzyme-substrate. Although the present invention relates generally to antigen-antibody type reactions, it also involves small molecules that bind antigens as defined herein.

As used herein, "treatment" includes all regimes that can benefit a human or non-human animal, preferably a mammal. Treatment may be directed to an existing condition or may be prophylactic (preventive treatment).

As used herein, a "tumor" is an abnormal growth of tissue. It may be localized (benign) or may invade nearby tissues (malignant) or distant tissues (metastatic). Tumors include neoplastic growths that cause cancer, as well as tumors of the esophagus, colorectal, stomach, breast, ovary, and endometrium, and cancerous tissues or cells, including but not limited to white blood cells. Including stocks. As used herein, "tumor" also includes endometriosis within its scope.

The term "antibody," as used herein, refers to immunoglobulin molecules and immunologically active immunoglobulin molecules, whether natural or partly or wholly synthetically produced. , i.e., a molecule that contains an antigen binding site that specifically binds to an antigen. The term also includes any polypeptide or protein having a binding domain that is or is homologous to an antibody binding domain. They may be derived from natural sources, or they may be partly or wholly synthetically produced. Examples of antibodies of the invention include immunoglobulin isotypes (eg, IgG, IgE, IgM, IgD, and IgA), and isotypic subclasses thereof; fragments comprising antigen binding domains, such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies can be polyclonal or monoclonal. A monoclonal antibody may be referred to as a "mAb."

It is possible to take monoclonal and other antibodies and use recombinant DNA technology to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding an immunoglobulin variable region or antibody CDRs into the constant region or constant plus framework regions of a different immunoglobulin. See for example EP-A-184187, GB-A-2188638 or EP-A-239400. A hybridoma or other cell that produces an antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.

As antibodies can be modified in many ways, the term "antibody" should be taken to encompass any specific binding member or substance having a binding domain with the requisite specificity. The term thus includes antibodies, antibody fragments, derivatives, functions of humanized antibodies, including all polypeptides containing immunoglobulin binding domains, whether natural or wholly or partially synthetic. equivalents, and homologues. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies is described in EP-A-0120694 and EP-A-0125023. A humanized antibody can be a modified antibody having a non-human, eg, murine, antibody variable region and a human antibody constant region. Methods for making humanized antibodies are described, for example, in US Pat. No. 5,225,539.

It has been shown that fragments of whole antibodies can perform the function of binding antigen. Examples of binding fragments are (i) a Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) the VL and (iv) a dAb fragment consisting of the VH domain (Ward, Gussow et al. 1989); (v) an isolated CDR region; (vi) a bivalent fragment comprising two linked Fab fragments. (vii) a single-chain Fv molecule (scFv), where the VH and VL domains form an antigen-binding site by being joined by a peptide linker that joins the two domains; (Bird, Hardman et al. 1988, Huston, Levinson et al. 1988); (viii) bispecific single-chain Fv dimers (WO 92/09965); and (ix ) "diabodies," multivalent or multispecific fragments constructed by gene fusion (WO 94/13804; (Holliger, Prospero et al. 1993)).

Diabodies are multimers of polypeptides, wherein each polypeptide comprises a first domain comprising the binding region of an immunoglobulin light chain and a second domain comprising the binding region of an immunoglobulin heavy chain; The two domains are linked (eg by a peptide linker) but cannot be linked together to form an antigen binding site: the antigen binding site is the first domain of one polypeptide in the multimer. Formed by association with a second domain of another polypeptide in a multimer (WO 94/13804).

Where bispecific antibodies are used, these may be conventional bispecific antibodies that can be produced in a variety of ways (Holliger and Winter 1993), for example prepared chemically or from hybrid hybridomas. or any of the bispecific antibody fragments described above. It may be preferable to use scFv dimers or diabodies over whole antibodies. Diabodies and scFv can be constructed using only variable domains and without an Fc region, possibly reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins" described in (Traunecker, Lanzavecchia et al. 1991).

Bispecific diabodies may also be useful because they can be readily constructed and expressed in E. coli, as opposed to bispecific whole antibodies. Diabodies (and many other polypeptides, such as antibody fragments) of appropriate binding specificity can be readily selected using phage display (WO 94/13804) from libraries. If one arm of the diabody remains constant, eg with specificity for antigen X, this creates a library in which the other arm is varied, and antibodies of appropriate specificity can be selected.

A "binding domain" is a portion of a specific binding member that includes a region that specifically binds to and is complementary to part or all of an antigen. When the binding member is an antibody or antigen-binding fragment thereof, the binding domains may be CDRs. If the antigen is large, the antibody may bind only a particular portion of the antigen, this portion is called the epitope. An antigen binding domain may be provided by one or more antibody variable domains. An antigen binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

"Specific" generally means that one member of a specific binding pair shows no significant binding to a molecule other than its specific binding partner, e.g. %, preferably less than 20%, less than 10%, or less than 1% cross-reactivity. The term is also applicable when, for example, an antigen binding domain is specific for a particular epitope carried by many antigens, in which case the specific binding member bearing the antigen binding domain bears the epitope can bind to a variety of antigens A specific binding member of the invention can be detected against any other antigen (e.g. any other glycan) when binding is tested by the protocol presented in the "Glycome Analysis" section of the Examples herein. It may be possible to specifically bind to Le ^y in the absence of possible binding.

"Isolated" refers to the situation in which a specific binding member of the invention or a nucleic acid encoding said binding member is preferably according to the invention. In general, members and nucleic acids are found in their natural environment or the environment in which they are prepared (when such preparation is by recombinant DNA technology performed in vitro or in vivo) (e.g., cell culture). are free or substantially free of materials with which they are naturally associated, such as polypeptides or nucleic acids of Specific binding members and nucleic acids may be formulated with diluents or adjuvants, and may also be isolated for practical purposes—for example, members are commonly prepared in microtiter plates for use in immunoassays. Mixed with gelatin or other carriers when used for coating, or with pharmaceutically acceptable carriers or diluents when used diagnostically or therapeutically. Specific binding members may be glycosylated, either naturally or by heterologous eukaryotic systems, or they may be non-glycosylated (eg when produced by expression in prokaryotic cells).

By "substantially as set out" is meant that the amino acid sequences of the invention are identical to or significantly homologous to the described amino acid sequence. By “significant homology” it is contemplated that 1-5, 1-4, 1-3, 2 or 1 substitutions can be made in the sequence.

The present invention also provides polypeptides having the amino acid sequences presented in Figures 1, 2, or 3, polynucleotides having the nucleic acid sequences presented in Figures 1 or 2, and substantial identity thereto, e.g. Sequences with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity are included within the scope. The percent identity of two amino acid sequences or two nucleic acid sequences is generally determined by aligning the sequences for optimal comparison (e.g. gaps are introduced into the first sequence for best alignment with the second sequence). obtained), determined by comparing the amino acid residues or nucleotides at corresponding positions. A "best alignment" is the alignment of two sequences that gives the highest percent identity. Percent identity is determined by comparing the number of identical amino acid residues or nucleotides in the sequences (ie, % identity = number of identical positions/total number of positions x 100).

The determination of percent identity between two sequences can be accomplished using mathematical algorithms known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990 (Karlin and Altschul 1990), modified as in Karlin and Altschul, 1993 (Karlin and Altschul 1993). Altschul et al. , 1990 (Altschul, Gish et al. 1990), the NBLAST and XBLAST programs incorporate such algorithms. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison, Gapped BLAST can be used as described in Altschul et al. , 1997 (Altschul, Madden et al. 1997). Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used. http://www. ncbi. nlm. nih. See gov. Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1989 (Myers and Miller 1989). The ALIGN program (version 2.0), part of the GCG sequence alignment software package, incorporates such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti, 1994 (Torelli and Robotti 1994); and Pearson and Lipman, 1988 (Pearson and Lipman 1988). and FASTA. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

An isolated specific binding member of the invention is capable of binding a Le ^y carbohydrate, which may be a Le ^y ceramide or may be a protein moiety. The binding domain comprising the amino acid sequence substantially as presented in residues 105-116 (CDRH3) of Figures 1a and 1b and 105-113 of Figure 1c is a structure that allows binding of these regions to Le ^y carbohydrates. can be carried.

The structures for carrying the binding domains of the invention are generally positioned so that the binding domains correspond to the CDR3 regions of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes. or a substantial portion thereof of an antibody heavy or light chain sequence located in the Structures and locations of immunoglobulin variable domains can be found at http://www. imgt. org/. The amino acid sequence substantially presented at residues 105-116 of Figures 1a and 1b may be carried as CDR3 of a human heavy chain variable domain or a substantial part thereof, residues 105-113 of Figure 1c. The amino acid sequence substantially presented as may be carried as CDR3 of a human light chain variable domain or a substantial part thereof.

The variable domains may be derived from any germline or rearranged human variable domains, or may be synthetic variable domains based on consensus sequences of known human variable domains. A CDR3-derived sequence of the invention can be introduced into a repertoire of variable domains lacking the CDR3 region using recombinant DNA technology. For example, Marks et al. , 1992 (Marks, Griffiths et al. 1992), for providing a repertoire of VH variable domains lacking CDR3, consensus primers directed to or flanking the 5' end of the variable domain region. describe a method for producing a repertoire of antibody variable domains used in conjunction with consensus primers to the third framework region of human VH genes. Further, Marks et al. , 1992 (Marks, Griffiths et al. 1992) describe how this repertoire can be combined with the CDR3 of a particular antibody. Using similar techniques, a CDR3-derived sequence of the invention may be shuffled with a repertoire of VH or VL domains lacking CDR3, and the shuffled complete VH or VL domain may be replaced with the cognate VL or VH Domains can be combined to provide specific binding members of the invention. This repertoire can then be displayed in a suitable host system, such as the phage display system of WO 92/01047, from which suitable specific binding members can be selected. A repertoire can consist of 10 ⁴ individual members or more, eg 10 ⁶ to 10 ⁸ or 10 ¹⁰ members.

Similar shuffling or combinatorial techniques are also disclosed by Stemmer, 1994 (Stemmer 1994), who describes techniques involving the β-lactamase gene, but this technique can be used to generate antibodies. I am observing. A further alternative is to generate novel VH or VL regions carrying sequences derived from the CDR3s of the invention using random mutagenesis, e.g. of the FG27 VH or VL gene, to create mutations in the entire variable domain. It is to make. Such techniques are described by Gram et al. using error-prone PCR. , 1992 (Gram, Marconi et al. 1992).

Another method that can be used is to generate mutagenesis to the CDR regions of the VH or VL gene. Such techniques are described in Barbas et al. , 1994 (Barbas, Hu et al. 1994) and Schier et al. , 1996 (Schier, McCall et al. 1996). A substantial portion of an immunoglobulin variable domain generally comprises at least three CDR regions along with their intervening framework regions. The portion can also comprise at least about 50% of either or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the fourth It is the N-terminal 50% of the framework region. Additional residues at the N-terminus or C-terminus of a substantial portion of the variable domain may not be normally associated with naturally occurring variable domain regions. For example, the construction of specific binding members of the invention, made by recombinant DNA techniques, may involve immunoglobulin heavy chains, other variable domains (eg, in the production of diabodies) or protein markers, as discussed in more detail below. N-terminal or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps, including the introduction of linkers to join the variable domains of the invention to additional protein sequences comprising can lead to the introduction of

The present invention provides binding domains based on the amino acid sequences of the VL and VH regions substantially as presented in Figure 1, namely amino acids 1-127 (VH) of Figure 1a or 1b and amino acids 1-124 (VL) of Figure 1c. Specific binding members are provided that include pairs of Single binding domains based on any of these sequences form further aspects of the invention. For binding domains based substantially on the amino acid sequences of the VH regions presented in FIGS. 1a and 1b, such binding domains are known to enable immunoglobulin VH domains to bind target antigens in a specific manner. Therefore, it can be used as a targeting agent. In the case of either single-chain specific binding domains, these domains are two-domain specific binding domains that have in vivo properties as good or comparable to the FG27 antibodies disclosed herein. can be used to screen for complementary domains capable of forming binding members.

This is a phage display screening method using the so-called hierarchical dual combinatorial approach disclosed in WO 92/01047, where either H or L chain clones was used to infect a complete library of clones encoding the other strand (L or H), and the resulting double-stranded specific binding members were identified in this reference. selected by phage display technology such as those disclosed). This technique is also described in Marks et al. , 1992 (Marks, Griffiths et al. 1992).

A specific binding member of the invention may further comprise an antibody constant region or part thereof. For example, specific binding members based on the VL region shown in Figure 1c may bind at their C-terminus to antibody light chain constant domains. Similarly, the specific binding members based on the VH regions shown in FIGS. 1a and 1b may have, at their C-terminus, any antibody isotype, e.g., IgG, IgA, IgE, and IgM, and isotype subclasses, In particular, it can bind all or part of immunoglobulin heavy chains derived from IgG1, IgG2, and IgG4.

Specific binding members of the invention may be used in methods of diagnosis and treatment of tumors in human or animal subjects.

When used for diagnosis, specific binding members of the invention may be labeled with a detectable label, for example a radiolabel such as ¹³¹ I or ⁹⁹ Tc, which are known in the art of antibody imaging. Specific binding members of the invention can be conjugated using conventional chemical techniques. Labels also include enzymatic labels such as horseradish peroxidase. Labels also include chemical moieties such as biotin, which can be detected via binding to certain cognate detectable moieties, eg, labeled avidin.

Although the specific binding members of the invention have been shown to be effective in killing cancer cells themselves, they can be further labeled with a functional label. Functional labels include substances designed to target cancer sites to effect destruction of the cancer cells. Such functional labels include toxins such as ricin and enzymes such as bacterial carboxypeptidase or nitroreductase, which can convert prodrugs into active drugs. In addition, specific binding members are chemotherapeutic or cytotoxic agents such as maytansines (DM1 and DM4), onides, auristatins, calicheamicins, duocamycins, doxorubicin, or ⁹⁰ Y or It may be attached to or otherwise associated with a radiolabel such as ¹³¹ I.

Furthermore, specific binding members of the invention may be administered alone or in combination with other treatments, either simultaneously or sequentially depending on the condition being treated. The invention thus further provides products comprising a specific binding member of the invention and an active agent as a combination formulation for simultaneous, separate or sequential use in the treatment of tumors. Effective agents may include chemotherapeutic or cytotoxic agents, including 5-fluorouracil, cisplatin, mitomycin C, oxaliplatin, and tamoxifen, which may act synergistically with the binding members of the invention. Other effective agents may include appropriate doses of analgesics, such as non-steroidal anti-inflammatory drugs (eg aspirin, paracetamol, ibuprofen, or ketoprofen) or opiates, such as morphine, or antiemetics.

Without wishing to be bound by theory, the property of binding members of the invention to act synergistically with effective agents to enhance tumor killing may not be due to immune effector mechanisms; Rather, it may be a direct result of binding of the binding member to Le ^y glycans bound to the cell surface. Cancer immunotherapy involving antibodies against immune checkpoint molecules has been shown to be effective in combination with different immuno-oncology treatment modalities against a variety of malignancies.

A specific binding member of the invention is usually administered in the form of a pharmaceutical composition, which may contain at least one component in addition to the specific binding member. Pharmaceutical compositions can include, in addition to the active ingredient, pharmaceutically acceptable excipients, diluents, carriers, buffers, stabilizers, or other substances well known to those of skill in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which can be oral or by injection, eg, intravenous injection. Although injection is the primary route for therapeutic administration of compositions, it is envisioned that delivery via catheters or other surgical tubes will also be used. Some suitable routes of administration include intravenous, subcutaneous, intraperitoneal, and intramuscular administration. Liquid formulations may be utilized after reconstitution from powder formulations.

For intravenous injection or injection at the affected site, the active ingredient is in the form of a pyrogen-free, parenterally acceptable aqueous solution having suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder, or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline solution, dextrose or other sugar solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. If the formulation is liquid, it may be, for example, a physiological salt solution or a lyophilized powder containing a non-phosphate buffer of pH 6.8-7.6.

The compositions may also be administered via microspheres, liposomes, other microparticle delivery systems or sustained release formulations that are placed in specific tissues, including blood. Suitable examples of sustained release carriers include semipermeable polymer matrices in common article forms such as suppositories or microcapsules. Polylactide (U.S. Pat. No. 3,773,919; European Patent Publication No. 0058481), a copolymer of L-glutamic acid and γ-ethyl-L-glutamate (Sidman, Steber et al. al. 1983), poly(2-hydroxyethyl-methacrylate). Liposomes containing polypeptides can be prepared by well-known methods: DE 3,218, 121A; (Eppstein, Marsh et al. 1985); (Hwang, Luk et al. 1980); EP 0088046; EP 0143949; EP 0142541; EP 83-11808; No. 4,544,545. Basically, liposomes are of the small (about 200-800 Angstroms) unilamellar type, in which the lipid content is greater than about 30 mol % cholesterol, and a selected proportion reduces polypeptide leakage. Adjusted for optimal proportions. The compositions may be administered in a localized manner to the tumor site or other desired site, or delivered in a manner that targets the tumor or other cells.

The compositions are preferably administered to an individual in a "therapeutically effective amount," which is an amount sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will vary depending on the nature and severity of what is being treated. Prescription of treatment, e.g., determination of dosage, is within the responsibility of general practitioners and other medical doctors and usually depends on the disorder being treated, the individual patient's condition, the site of delivery, the method of administration, and what is known to the practitioner. Consider other factors that The compositions of the invention are particularly relevant for the treatment of existing tumors, especially cancer, and the prevention of recurrence of the condition after initial treatment or surgery. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, ^16th edition, Oslo, A.; (ed), 1980 (Remington 1980).

Optimal dosages can be determined by a physician based on a number of parameters including, for example, age, sex, weight, severity of condition being treated, active ingredient administered, and route of administration. In general, serum concentrations of polypeptides and antibodies that allow saturation of the receptor are desirable. Concentrations above about 0.1 nM are usually sufficient. For example, a dose of 100 mg/m ² of antibody provides a serum concentration of about 20 nM for about 8 days.

As a rough guideline, antibody doses may be administered weekly in amounts of 10-300 mg/m ² . Equal doses of antibody fragments are used at more frequent intervals to maintain serum levels above those that allow saturation of ^Ley carbohydrates. The dosage of the composition depends on the properties of the binding member, such as its binding activity and in vivo plasma half-life, concentration of the polypeptide in the formulation, route of administration, site and rate of administration, clinical tolerance of the patient involved, It varies according to the condition afflicting the patient and is within the skill of the physician. For example, although a dose of 300 μg of antibody per patient per dose is preferred, doses can range from about 10 μg to 6 mg per dose. Different doses are utilized during a series of sequential inoculations. Practitioners can give an initial inoculation followed by a boost with relatively low doses of antibody.

The present invention is also directed to optimizing immunization schedules to enhance protective immune responses against cancer.

Binding members of the invention may be made by chemical synthesis, in whole or in part. Binding members can be readily prepared by well-established standard solution-phase or preferably solid-phase peptide synthesis methods (this general description is widely available (e.g. JM Stewart and J. D. Young, 1984 (Stewart and Young 1984), M. Bodanzsky and A. Bodanzsky, 1984 (Bodanzsky and Bodanzsky 1984)), or which, in solution, use liquid-phase methods or solid-phase, liquid-phase, and By any combination of solution chemistry techniques, for example, each peptide moiety is first completed and then, if desired and appropriate, after removal of any protecting groups present, each carbonic acid or sulfonic acid or their reactive can be prepared by introducing a residue X by reaction of a derivative of

Another convenient way of producing a binding member according to the invention is to express the nucleic acid encoding it by using the nucleic acid in an expression system.

The invention further provides isolated nucleic acids encoding specific binding members of the invention. Nucleic acids include DNA and RNA. In a preferred aspect, the invention provides nucleic acids encoding the specific binding members of the invention as defined above. Examples of such nucleic acids are shown in FIGS. One skilled in the art can determine substitutions, deletions and/or additions to the nucleic acid that still provide specific binding members of the invention.

The invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes comprising at least one nucleic acid as described above. The invention also provides recombinant host cells containing one or more of the constructs described above. As noted, nucleic acids encoding specific binding members of the invention form an aspect of the invention, as do methods of producing specific binding members, including expression from the encoding nucleic acids. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. After production by expression, the specific binding member may be isolated and/or purified using any suitable technique and then used as appropriate.

Systems for cloning and expression of polypeptides in a variety of different host cells are well known. Suitable host cells include bacterial, mammalian cells, yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, and many others. A common preferred bacterial host is E. coli. Expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art. For a review see, for example, Pluckthun, 1991 (Pluckthun 1991). Eukaryotic expression in culture is also available to those skilled in the art as an option for the production of specific binding members. Recent reviews include, for example, Reff, 1993 (Reff 1993); Trill et al. , 1995 (Trill, Shatzman et al. 1995).

Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, transcription termination sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral eg phage, or phagemid, as appropriate. For further details see, for example, Sambrook et al. , 1989 (Sambrook 1989). Many known techniques and protocols for manipulation of nucleic acids, mutagenesis, sequencing, introduction of DNA into cells, and gene expression, for example, in the preparation of nucleic acid constructs, and analysis of proteins are described in Ausubel et al. , 1992 (Ausubel 1992).

A further aspect of the invention thus provides a host cell comprising a nucleic acid disclosed herein. A further aspect provides a method comprising introducing said nucleic acid into a host cell. This introduction may use any available technique. In eukaryotic cells, suitable techniques include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and retroviruses or other viruses such as vaccinia virus, or, in insect cells, baculovirus. can include transduction of For bacterial cells, suitable techniques may include transformation with calcium chloride, electroporation, and transfection using bacteriophage. After this introduction, expression from the nucleic acid may be induced or allowed, for example by culturing the host cell under conditions for gene expression.

A nucleic acid of the invention can be integrated into the genome (eg, chromosome) of the host cell. Integration can be facilitated by the inclusion of sequences that facilitate recombination in the genome by standard techniques.

The invention also provides methods comprising using the above constructs in an expression system to express the specific binding members or polypeptides described above.

Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis. The prior art documents referred to herein are incorporated to the fullest extent permitted by law.

Unexpectedly, we found that the ^FG27 family of mAbs that recognize Ley-containing glycans induced non-apoptotic direct cell death without involving immune effector cells. In one aspect of the invention, the invention provides drugs that bind to ^Ley -containing glycans and induce non-apoptotic cell death.

The FG27 mAb induced membrane damage to the cells, resulting in cell clumping, microvillus loss, small molecular weight dye uptake and pore formation. Over time, the cells developed large pores and lysed by a mechanism similar to necrosis. This is similar to many mAbs that recognize other glycans (Hellstrom, Garrigues et al. 1990, Chou, Takematsu et al. 1998, Zhong, Manzi et al. 2001, Alvarez-Rueda, Leprieur et al. 2007 , Loo, Pryer et al. 2007, Zhang, Zhang et al. 2010). FG27 mAb also exhibited potent cytotoxic activity in vitro via ADCC and CDC. Administration of FG27 (0.1 mg intravenously (i.v.) twice weekly for 9 weeks) to mice with established metastases in the liver and peritoneal cavity resulted in complete tumor eradication and approximately Resulted in 30% healing. The potential of ^FG27 mAbs in eradicating well-established tumors without concurrent chemotherapy suggests their potential as monotherapeutic agents for the treatment of Ley-expressing human solid tumors. represent.

The invention will now be further illustrated by the following non-limiting examples and the accompanying drawings.

Figure 1a: Complete amino acid and nucleotide sequence of mouse FG27.10 IgG3 heavy chain. Numbers represent the standardized IMGT system for antibody sequence numbering (Lefranc, Giudicelli et al. 2009). Figure 1b: Complete amino acid and nucleotide sequence of mouse FG27.18 IgG1 heavy chain. Numbers represent the standardized IMGT system for antibody sequence numbering (Lefranc, Giudicelli et al. 2009). Figure Ic: Complete amino acid and nucleotide sequences of the kappa chains of mouse FG27.10 and FG27.18. Numbers represent the standardized IMGT system for antibody sequence numbering (Lefranc, Giudicelli et al. 2009). Ditto. Ditto. Figure 2a: Complete amino acid and nucleotide sequences of mouse FG27 heavy chain variable region chimerized with human IgG1 heavy chain constant region. Numbers represent the standardized IMGT system for antibody sequence numbering (Lefranc, Giudicelli et al. 2009). Figure 2b: Complete amino acid and nucleotide sequences of mouse FG27 kappa chain variable region chimerized with human kappa chain constant region. Numbers represent the standardized IMGT system for antibody sequence numbering (Lefranc, Giudicelli et al. 2009). Ditto. Figure 3a: Complete amino acid and nucleotide sequences of the humanized FG27 heavy chain variable region. Numbers represent the standardized IMGT system for antibody sequence numbering (Lefranc, Giudicelli et al. 2009). Figure 3b: Complete amino acid and nucleotide sequences of the humanized FG27 kappa chain variable region. Numbers represent the standardized IMGT system for antibody sequence numbering (Lefranc, Giudicelli et al. 2009). Ditto. Figure 4: Binding of FG27.10 and FG27.18 to ST16, HUVEC and PBMC. Negative controls for this assay were NSO supernatants for ST16 and HUVECs, or cells alone for PBMCs, and positive controls were anti-sialyl-diLe ^a mAb, 505/4, or W6/ was 32. Figure 5: FG27.10 and FG27.18 on glycolipid extracts from colorectal (C170), ovarian (OVCAR-3, OVCAR-4, OVCA433, OAW28, OAW42) and gastric cell lines (ST16, MKN45, AGS) binding. Glycolipids extracted from a range of cancer cell lines were plated on ELISA plates. Cells and glycolipids were then incubated with FG27.10 or FG27.18 supernatants. Binding to glycolipids was probed with anti-IgG-HRP/TMB. Figure 6: Binding of FG27.10 to HSA-coupled Lewis antigen (a, b, x, and y) assessed by ELISA. 505/4 (anti-sialyl-di-Le ^a ), FG88.2 (Le ^ax ), FG88.7 (Le ^ax ), and 225-Le (Le ^b ) were included as positive controls and mouse HRPO was included as a negative control. Antibody activity was measured by absorbance at 405 nm. Figure 7: Binding of FG27.10 and FG27.18 was screened against The Consortium for Functional Glycans glycan array composed of 610 mammalian glycan targets. The fine specificity between a) FG27.10, b) FG27.18, c) 692 (Le ^y/b ), and d) BR96 (Le ^y/x ) was compared: where a=Le ^a , b=Le ^b , y=Le ^y , x-Le ^x , D-=di, T-=tri, S-=sialyl, Ex-=extended, φ represents mannose containing glycans, yx=Le ^y -Le ^x . Note that the glycan number may not be the same for each mAb as the mAbs were screened on different versions of the glycan array. Ditto. Figure 8: Binding of ^FG27 to Ley bound to total protein or lipids extracted from a panel of tumor cell lines. FG27 against Le ^y associated total glycoproteins or glycolipids extracted from tumor cell lines, AGS cells, HCT15 cells, OVCAR3 cells, MCF7 cells, and H322 cell lysates (equivalent to 1×10 ⁵ cells). Detection of binding by Western blot. Figure 9: Comparison of anti-Le ^y and Le ^b mAbs on a range of cell lines. SC101 (692/29) and BR96 were titrated at 0.1-30 μg/ml across a range of cell lines (ST16, HT29, C170, LoVo, Colo201, OVCAR-3, AGS), where Clears were diluted 1:30 from neat. Binding was probed with anti-IgG-FITC and analyzed by flow cytometry. Figure 10: Representative normal esophagus, stomach, duodenum, ileum, colon, pancreas, lung, and kidney stained with 692/29 (Le ^y/b ), BR96 (Le ^y/x ), and FG27 (Le ^y ). typical IHC image. Figure 11a: In vivo anti-tumor activity of FG27. The percentage of tumor growth over the study period is shown in the C170HM2 bioluminescent mouse tumor model used to assess the anti-tumor activity of mouse FG27 mAb compared to vehicle only control (PBS). In this model, bioluminescence represents viability. Groups n≧9; the study was terminated on day 32. Treatment with FG27 resulted in a significant reduction in bioluminescent tumor burden by the last day of study compared to vehicle controls (p=0.014). FIG. 11b: Injection site tumor burden as assessed by BLI shows a significant reduction in injection site tumor growth with FG27 treatment (p=0.05). Figure 12: In vivo anti-tumor activity of FG27. Percentage of tumor growth is shown in the C170HM2 bioluminescent mouse tumor model used to assess the anti-tumor activity of FG27 compared to positive control mAb and vehicle only control (PBS). Bioluminescence represents tumor cell viability in this model. Groups n≧8; treatment with FG27 resulted in a significant reduction in the percentage of tumor growth by day 51 compared to treated controls (p=0.009). Figure 12b: Analysis by log-rank Mantel-Cox test reveals significant survival of the FG27-treated group compared to vehicle-only controls (p=0.035). Treatment ended on day 120. Figure 13a: Mouse FG27 heavy chain variable region amino acid and nucleotide sequences compared to BR96, H18A, Hu3S193, and SC101 using the standardized IMGT system for antibody sequence numbering (Lefranc, Giudicelli et al. 2009). Figure 13b: Amino acid and nucleotide sequences of mouse FG27 kappa chain variable regions compared to BR96, SC101, H18A, Hu3S193 using the standardized IMGT system for antibody sequence numbering (Lefranc, Giudicelli et al. 2009). Ditto. Ditto. Ditto. FIG. 14: Binding of chimeric IgG1 (5 μg/ml) and IgG2 (5 μg/ml) mAbs to AGS of ^Ley -expressing tumor cell lines as assessed by indirect immunofluorescence and flow cytometry analysis. Mouse versions of FG27.10 (5 μg/ml) and FG27.18 (5 μg/ml) were compared. Anti-HLA mAb W6/32 (1/100) was included as a positive control. Figure 15a: PI uptake in ST16 cells mediated by mouse IgG3 mAb FG27.10 induced strong PI uptake even at 4°C, but the IgGl variant FG27.18 did not. Figure 15b: FG27.10- and FG27.18-mediated PI uptake in a panel of tumor cell lines (AGS, Colo201, C170, C170HM2, MCF7, OVCA4, LoVo, MKN45, 791T, and Skov3). Figure 15c: PI uptake mediated by FG27.10, FG27.18, CH27 IgG2, CH27 IgGl in C170 cells. Ditto. Figure 16: Data for internalization of FG27.10, FG27.18, and CH27 over a range of concentrations (0.01 nM to 30 nM) in a panel of cancer cell lines, AGS, MCF7, H322, and C170. Internalization of the mAbs in cells was monitored with goat antibody against saporin-conjugated mouse IgG (ATSbio, IT#48) or goat antibody against saporin-conjugated human IgG (ZAP) (F'ab fragment) 75 ng/well. and assessed by inhibition of thymidine incorporation (added during the last 24 hours of incubation). Control wells consisted of cells incubated with culture medium alone, an irrelevant positive control IgG3 mAb FG88, and secondary conjugate alone. Figure 17a: Example of FG27.10 mIgG3 mediating ADCC/CDC. Target cells were incubated with FG27.10 supernatant or PBMC isolated from human blood (ADCC) or complement (CDC) with irrelevant IgG. Error bars representing the standard deviation of triplicate wells are present and may be ambiguous depending on the data point. Figure 17b: ADCC of MCF7 tumor cell lines upon incubation with FG27.10, FG27.18, 692/29, CH27 IgG1, and CH27 IgG2. These are results from one experiment, but represent two replicates. Figure 17c: Mean percentage killing of ST16 tumor cell lines by CDC after incubation with FG27.10, FG27.18, 692/29, CH27 IgG1, and CH27 IgG2 with human serum after 4 hours. Herceptin was included as a control. The assay was performed with serial dilutions of 3, 1, 0.3, and 0.1 μg/ml and each dilution was incubated in triplicate. Figure 17d: Mean percentage killing of AGS cells by CDC after incubation with FG27.10, 692/29, CH27 IgG1, and CH27 IgG2 with human serum after 4 hours. The assay was performed with serial dilutions of 3, 1, 0.3, and 0.1 μg/ml and each dilution was incubated in triplicate. Ditto. Ditto. Figure 18: Gibbs Analysis I of FG27 heavy chain: sequence identity. Structurally important residues are highlighted: proline residues are outlined in white, cysteine residues are outlined in black, and asparagine residues are outlined in grey. . Figure 19: Gibbs Analysis II of FG27 heavy chain: identity and similarity scores, 4 Å proximal residues and loop lengths of CDRs. Cysteine residues are outlined in white. Figure 20: Human heavy chain donor sequence germline analysis. Figure 21: The structurally important residues of the FG27 heavy chain humanization strategy are highlighted: proline residues are outlined in white, cysteine residues are outlined in black, and asparagine residues. The base is outlined in grey. Residues outlined in gray and in italics represent back-translations to murine residues. Figure 22: Gibbs Analysis I of 27 kappa light chains: sequence identity. Structurally important residues are highlighted: proline residues are outlined in white, cysteine residues are outlined in black, and asparagine residues are outlined in grey. . Figure 23: Gibbs Analysis II of FG27 kappa light chain: identity and similarity scores, 4 Å proximal residues and loop lengths of CDRs. Figure 24: Human kappa light chain donor sequence germline analysis. Figure 25: FG27 kappa light chain humanization strategy. Structurally important residues are highlighted: proline residues are outlined in white and cysteine residues are outlined in black. Residues in bold represent back-translation to mouse residues. Figure 26: Lewis Y-HSA antigen in combinations of antibodies designated as LewRHA and LewRKA, LewRKB, LewRKC, or LewRKD, and LewRHB and LewRKA, LewRKB, LewRKC, or LewRKD, including cFG27 positive control antibody and c1210 negative control antibody. Comparison of antibody binding to Figure 27. Thermal stability of LewRHA and LewRKA, LewRKB, LewRKC, or LewRKD, and LewRHB and LewRKA, LewRKB, LewRKC, or LewRKD with cFG27 positive control antibody. FIG. 28. Analysis of thermal shifts of Lewis ^y FG27 humanized variants hLewAB, hLewAC, hLewAD, hLewBC and control mouse FG27. FIG. 29. BiaCore analysis of the affinity of antibodies for FG27 humanized variants hLewAB, hLewAC, hLewAD, hLewBC and control mouse FG27. FIG. 30. Non-specific protein-protein interactions with FG27 humanized variants hLewAB, hLewAC, hLewAD, hLewBC and control mouse FG27 as measured by CIC (Cross-Interaction Chromatography). FIG. 31. Binding of FG27.10, FG27.18, ch27IgG1, and humanized variant hLewAC to a panel of cell lines. Figure 32: Significant increase in growth inhibition on MCF7 (panel i) and AGS (panel ii) by i27G1 compared to 27hIgG1. Significant increase in functional affinity (SPR) by i27G1 compared to 27hIgG1 (panel iii). i27G1 maintains comparable ADCC (panel iv) and CDC (panel v) activity with MCF7. Significance compared to parental constructs was estimated from two-way ANOVA (direct cytotoxicity) or one-way ANOVA (functional affinity) with Dunnett's correction for multiple comparisons. Figure 33: Amino acid and nucleic acid sequences of each light and heavy chain of the "i27G1" antibody.

Methods Binding to Tumor Cell Lines: 1 x 105 cancer cells were incubated with ⁵⁰ μl of primary antibody for 1 hour at 4°C. Cells were washed with 200 μl RPMI 10% new born calf serum (NBCS: Sigma, Poole, UK) and spun at 1,000 rpm for 5 minutes. The supernatant was discarded and 50 μl of FITC-conjugated anti-mouse IgG Fc-specific mAb (Sigma; 1/100 in RPMI 10% NBCS) was used as secondary antibody. Cells were incubated for 1 hour at 4° C. in the dark, then washed with 200 μl RPMI 10% NBCS and spun at 1,000 rpm for 5 minutes. After discarding the supernatant, cells were fixed using 0.4% formaldehyde. Samples were analyzed on a Beckman Coulter FC-500 flow cytometer (Beckman Coulter, High Wycombe, UK). WinMDI 2.9 software was used to analyze and plot the raw data.

Binding to blood: 50 μl of healthy donor blood was incubated with 50 μl of primary antibody for 1 hour at 4°C. Blood was washed with 150 μl of RPMI 10% NBCS and spun at 1,000 rpm for 5 minutes. The supernatant was discarded and 50 μl of FITC-conjugated anti-mouse IgG Fc-specific mAb (1/100 in RPMI 10% NBCS) was used as secondary antibody. Cells were incubated for 1 hour at 4° C. in the dark, then washed with 150 μl RPMI 10% NBCS and spun at 1,000 rpm for 5 minutes. After discarding the supernatant, red blood cells were lysed using 50 μl/well Cal-Lyse (Invitrogen, Paisley, UK) followed by 500 μl/well distilled water. The blood was then spun at 1,000 rpm for 5 minutes. Supernatant was discarded and cells were fixed using 0.4% formaldehyde. Samples were analyzed on an FC-500 flow cytometer (Beckman Coulter). WinMDI 2.9 software was used to analyze and plot the raw data.

Extraction of Cell Membrane Glycolipids: Cell pellets (5×10 ⁷ cells) were resuspended in 500 μl of mannitol/HEPES buffer (50 mM mannitol, 5 mM HEPES, pH 7.2, both Sigma) and 30 The pulse was passed through 3 needles (23G, 25G, 27G). 5 μl of 1 M CaCl ₂ was added to the cells and passed through 3 needles with 30 pulses each as above. The sheared cells were incubated on ice for 20 minutes and then spun at 3,000 g for 15 minutes at room temperature (RT). The supernatant was collected and spun at 48,000 g for 30 minutes at 4° C. and the supernatant was discarded. The pellet was resuspended in 1 ml methanol followed by 1 ml chloroform and incubated for 30 min at RT with rotation. The sample was then spun at 1,200 g for 10 minutes to remove precipitated protein. Supernatants containing cell membrane glycolipids were collected and stored at -20°C.

Glycome Analysis: To clarify the fine specificity of the FG27 mAb, the antibody was FITC-labeled and analyzed by the Consortium for Functional Glycomics (http://www.functionalglycomics.org/static/consortium/resources/resourcecoreh8.shtml). sent to. Here they were screened against over 600 natural and synthetic glycans. Briefly, synthetic and mammalian glycans with amino linkers are printed on glass microscope slides that are activated with N-hydroxysuccinimide (NHS) to form amide bonds. This work was performed by Core H of CFG at Emory University. FG27 was tested at room temperature at 1 μg/ml in PBS. Briefly, antibody samples were applied to the printed surface of the microarray and incubated for 1 hour in a humidified chamber. The slides were then rinsed four times with PBS before adding fluorescently labeled (Alexa Fluor 488) anti-rabbit IgG and incubating for 1 hour. The slides were then rinsed four times with PBS and fluorescence was measured with a Perkin-Elmer microarray XL4000 scanner and analyzed using Imagene software (BioDiscovery).

Further details of the protocol used are described below.

Glycan binding assay using unlabeled monoclonal antibody 1 . introduction:
1.1. The primary goal of Core H is to determine the Glycan Binding Protein (GBP) and binding specificities of various organisms submitted by researchers using printed glycan microarrays.

2. Reference address:
2.1. www. functional glycomics. org

3. Substances required:
3.1. Slides with glycans printed on the side of the slide with white etched barcodes and black marks (Core D) - do not touch this area.
3.2. Cover glass (Fisher scientific, 12-545F)
3.3. A wet slide processing chamber (Fisher scientific, NC9091416), or a home-made system using Petri dishes; here with a wet paper towel on the bottom of the chamber.
3.4. 100 ml Coplin jar for washing slides 3.5. Tris-HCl (Fisher scientific, BP152-1)
3.6. NaCl (Fisher scientific, S271-3)
3.7. CaCl2 (Fisher scientific, C79-500)
3.8. MgCl2 (Fisher scientific, BP214-500)
3.9. Potassium dihydrogen phosphate (Fisher scientific, P285-3)
3.10. dH20
3.11. Cyanine 5-streptavidin (ZYMED 43-4316)
3.12. Appropriate secondary antibody, fluorescently labeled when possible.
3.13. BSA (Fisher scientific, Bp1600-100)
3.14. Tween-20 (EMD Biosciences, 655205)
3.15. Sodium azide (fisher scientific, S227-500)
3.16. ProScan Array Scanner (Perkin Elmer)

4. buffer:
4.1. TSM = 20 mM Tris-HCl, pH 7.4 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2
4.2. TSM Wash Buffer (TSMW) = TSM Buffer + 0.05% Tween-20
4.3. TSM Binding Buffer (TSMBB) = TSM buffer + 0.05% Tween 20 + 1% BSA

5. protocol:
5.1. Make working stocks of wash buffers (TSM, TSM Wash Buffer, and H2O) or collect reagents and bring to room temperature if refrigerated.
5.1.1. Buffer (A) TSM-20 mM Tris-HCl, pH 7.4 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2
5.1.2. Buffer (B) TSM Wash Buffer (TSMW) - TSM Buffer + 0.05% Tween-20
5.1.3. Buffer (C) TSM Binding Buffer (TSMBB) - TSM buffer + 0.05% Tween 20 + 1% BSA
5.1.4. dH2O
5.2. Prepare a 100 μl sample by diluting the antibody in TSMBB or an appropriate binding buffer to a final concentration of 5-50 μg/ml or the appropriate concentration required for analysis based on antibody properties.
5.3. Remove the slide from the desiccator and label the sample name near the bar code outside the black mark on the slide.
5.4. Hydrate the slides by placing them in a glass Coplin staining jar containing 100 ml of TSMW for 5 minutes.
5.5. Remove excess liquid from the slide by holding the slide upright and draining the liquid.
5.6. Carefully apply 70 μl of sample (see 5.2) near the leftmost slide between the black marks.
5.7. Gently place the coverslip onto the slide to prevent bubbles from forming on the sample under the coverslip. If necessary, remove bubbles by gently tapping the coverslip with a pipette tip or gently lifting one side of the coverslip. Make sure the coverslip is between the black marks.

SDS-PAGE and Western Blot Analysis: Briefly, 10 ⁵ or 10 ⁶ cell equivalents (cell lysates, cell membrane lipid extracts) were analyzed for FG27 binding. Tumor cell membrane lipid extracts and cell lysates were reduced with dithiothreitol (DTT; Pierce Biotechnology, ThermoFisher, Loughborough, UK) using NOVEX 4%-12% Bis-Tris gels (Invitrogen). Subjected to SDS-PAGE and transferred to Hybond-P PVDF membrane (GE Healthcare, Amersham, UK) using 1× transfer buffer (20×, Invitrogen) and 20 (v/v)% methanol for 1 hour at 30V. . Membranes were blocked with 5 (w/v)% non-fat dry milk in 0.05 (v/v) Tween-PBS for 1 hour, then primary diluted in Tween-PBS, 2% BSA. Probed with antibody for 1 hour. Primary antibody binding was detected for 1 hour using a biotin-conjugated anti-mouse IgG Fc-specific secondary antibody (Sigma; 1/2000 dilution in Tween-PBS, 2% BSA) for 1 hour. Visualization was performed using 800 CW streptavidin (LICOR Biosciences, UK; 1/1000 in Tween-PBS 2% BSA).

Immunohistochemistry Evaluation of FG27: To determine the therapeutic value of FG27, it was evaluated by immunohistochemistry (IHC) on gastric, colorectal, ovarian, and breast tumor tissue microarrays. Screened.

Methods: Immunohistochemistry was performed using standard avidin-biotin peroxidase methods. Paraffin-embedded tissue sections were placed on a block heated to 60° C. to melt the paraffin. Tissue sections were deparaffinized with xylene and rehydrated via graded alcohols. The sections were then immersed in 500 ml of citrate buffer (pH 6) and heated in a microwave (Whirlpool) for 20 minutes to retrieve the antigen. Endogenous peroxidase activity was blocked by incubating tissue sections with endogenous peroxidase solution (Dako Ltd, Ely, UK) for 5 minutes. Normal pig serum (NSS; Vector Labs, CA, USA; 1/50 PBS) was added to each section for 20 minutes to block non-specific primary antibody binding. All sections were incubated with Avidin D/Biotin blocking kit (Vector Lab) for 15 minutes to block non-specific binding of avidin and biotin, respectively. Sections were reblocked with NSS (1/50 PBS) for 5 minutes. Tissue sections were then incubated with primary antibody for 1 hour at RT, anti-β-2-microglobulin (Dako Ltd; 1/100 in PBS) mAb and PBS alone were used as positive and negative controls, respectively. Tissue sections were washed with PBS and incubated with biotinylated goat anti-mouse/rabbit immunoglobulins (Vector Labs; 1/50 in NSS) for 30 min at RT. Tissue sections were washed with PBS and incubated with preformed 1/50 (PBS) streptavidin-biotin/horseradish peroxidase complex (Dako Ltd) for 30 min at RT. 3,3'-Diaminobenzidine tetrahydrochloride (3,3'-Diaminobenzidine tetrahydrochloride: DAB) was used as substrate. Each section was incubated twice with 100 μl of DAB solution for 5 minutes. Finally, sections were lightly counterstained with hematoxylin (Sigma-Aldrich, Poole Dorset, UK), dehydrated in graded alcohols, washed by soaking in xylene, and mounted in DPX (Distyrene, plasticiser, xylene). The slides were mounted using the agent (Sigma).

Identification of heavy and light chain variable regions of ^FG27.10 and FG27.18. Harvested and washed once with PBS, the cell pellet was treated with 500 μl of Trizol (Invitrogen). After dispersing the cells in the reagent, they were stored at −80° C. until RNA was prepared according to the manufacturer's protocol. RNA concentration and purity were determined by Nanodrop. Prior to cDNA synthesis, RNA was treated with DNase I to decontaminate genomic DNA according to the manufacturer's recommendations (DNase I recombinant, RNase-free, Roche Diagnostics, Burgess Hill, UK).

Synthesis of cDNA: First strand cDNA was prepared from 3 μg of total RNA using the first strand cDNA synthesis kit and AMV reverse transcriptase according to the manufacturer's protocol (Roche Diagnostics). After cDNA synthesis, reverse transcriptase activity was destroyed by incubation at 90°C for 10 minutes and the cDNA was stored at -20°C.

GAPDH PCR to assess cDNA quality: PCR was used to assess cDNA quality; primers specific for the mouse GAPDH housekeeping gene (5'-TTAGCACCCCTGGCCAAGG-3' and ) was used with hot-start Taq polymerase (NEB, Hitchen, UK) for 35 cycles (95° C., 3 min, then 35 cycles of 94° C./30 sec, 55° C./30 sec, 72° C./1 min: final finishing step at 72° C. for 10 minutes). Amplified products were evaluated by agarose gel electrophoresis.

PCR primer design for cloning of FG27 variable regions: Primers were designed to amplify the heavy and light chain variable regions based on the sequence data of the PCR products. Primers were designed to allow cloning of the relevant strands into unique restriction sites in the hIgG1/κ dual expression vector pDCOrig-hIgG1. Each 5' primer is targeted to a defined variable region start codon and leader peptide, with the Kozak consensus immediately 5' of the start codon. Each 3' primer should be complementary to the binding region of the antibody sequence, should maintain the reading frame after strand cloning, and should preserve the amino acid sequence normally found at the binding/constant region junction. , designed. All primers were purchased from Eurofins MWG.

Heavy Chain Variable Region PCR: Immunoglobulin heavy chain variable region usage was determined using PCR with previously published primer sets (Jones and Bendig 1991). Previous results using a mouse mAb isotyping test kit (Serotec, Oxford, UK) indicate that FG27.10 is a mouse IgG3 antibody and FG27.18 is a mouse IgG1 antibody. Therefore, an appropriate constant region reverse primer was used to amplify from the constant region. PCR amplification was performed for 35 cycles (94°C for 5 min, then 35 cycles of 94°C/1 min, 60°C/1 min, 72°C/2 min; final finishing step at 72°C for 20 min). , with 5′ and 3′ primers specific to 12 mouse VH regions, specific to antibody subclasses previously determined using hot-start Taq polymerase. Amplified products were evaluated by agarose gel electrophoresis. Positive amplification was produced with VH4 primers.

PCR for light (κ) chain variable regions: Immunoglobulin light chain variable region usage was determined using PCR with previously published primer sets (Jones and Bendig 1991). Previous results using mouse mAb isotyping test kits indicate that both FG27.10 and FG27.18 used kappa light chains. PCR amplification was performed for 35 cycles (94°C for 5 min, then 35 cycles of 94°C/1 min, 60°C/1 min, 72°C/2 min; final finishing step at 72°C for 20 min). , with primers specific for the 5′ and 3′ mouse Cκ specific for the mouse Vκ region using hot-start Taq polymerase. Amplification products were evaluated by agarose gel electrophoresis. Positive amplification was produced by Vκ1 and Vκ2 primers in both FG27.10 and FG27.18.

Purification and sequencing of PCR products: PCR products were purified using the Qiaquick PCR purification kit (Qiagen, Crawley, UK). The concentration of DNA obtained was determined by Nanodrop and purity was assessed by agarose gel electrophoresis. The PCR product was analyzed at the University of Nottingham DNA sequencing facility (http://www.nottingham.ac.uk/life-sciences/facilities/dna-sequencing/index.aspx) using the original 5' and 3' PCR primers. was sequenced using Sequences were analyzed using the IMGT database search facility (http://www.imgt.org/IMGT_vquest/vquest?livret=0&Option=mouseIg) (identification of V regions, analysis of junctions). Sequencing showed that FG27.10 and FG27.18 share identical heavy and light chain variable regions. A constant region of sufficient residues is present in the heavy chain sequence that FG27.10 is a subclass of mIgG3 and FG27.18 is a subclass of mIgG1, the two resulting from a single splenocyte NSO fusion event. It was confirmed that

Cloning strategy: Direct cloning of PCR products into pDCOrig-hIgG1 using restriction sites built into the PCR primers was known to be relatively inefficient from previous Scancell experience. Therefore, a dual cloning strategy was employed: PCR products generated using a proofreading polymerase were cloned simultaneously into both pDCOrig-hIgG1 and TA vector (pCR2.1; Invitrogen), where TA The product cloned into the vector served as a back-up source of readily amplified material should the initial pDCOrig-hIgG1 cloning fail.

FG27.18 heavy/light chain PCR for cloning: Amplify PCR for 35 cycles (98°C for 3 min, then 35 cycles of 98°C/30 sec, 58°C/30 sec, 72°C/45 sec 72° C., 3 min final polishing step) using the FG27.18 cDNA template described above and the proofreading polymerase (Phusion; NEB) and cloning primers described above. Successful amplification was confirmed by agarose gel electrophoresis.

Method 1 - Direct Light Chain Cloning: The amplified FG27.18 light chain was sequentially digested with the restriction enzymes BsiWI and BamHI according to the manufacturer's instructions (NEB). A vector (pDCOrig-hIgG1 containing the V regions from the previously cloned antibody) was digested at the same time. Vector DNA was purified by agarose gel using a QIAquick gel extraction kit (Qiagen) and insert DNA was purified using a PCR purification kit. After DNA quantification by Nanodrop, the vector DNA was treated with phosphatase according to the manufacturer's recommendations (Antarctic Phosphatase, NEB) and the light chain insert was ligated into the vector (T4 DNA ligase, NEB). The ligated DNA was transformed into chemically competent TOP10F' cells (Invitrogen), plated on LB agar plates supplemented with 35 μg/ml Zeocin (Invitrogen, Toulouse, France) and then plated at 37°C. Incubated overnight.

Method 2 - TOPO light chain cloning: Amplified FG27.18 light chain was treated with Taq polymerase (NEB) for 15 minutes at 72°C to add 'A' overhangs compatible with TA cloning. Treated PCR products were incubated with TOPO TA vector pCR2.1 (Invitrogen) and transformed into chemically competent TOP10F' cells according to the manufacturer's instructions. Transformed bacteria were plated on LB agar plates supplemented with ampicillin (80 μg/ml) and then incubated overnight at 37°C. Colonies were grown in liquid culture (LB supplemented with 80 μg/ml ampicillin) and plasmid DNA was prepared (spin miniprep kit, Qiagen). The presence of the insert was confirmed by sequential digestion with BsiWI and BamHI and agarose gel electrophoresis. Sequencing was performed on miniprep DNA from colonies using T7 and M13rev primers. A DNA insert from one such colony has the expected FG27.18 light chain sequence; Plasmid maxi kit, Qiagen). The insert of Maxiprep DNA was confirmed by sequencing.

Cloning of TOPO heavy chain: Amplified FG27.18 heavy chain was treated with Taq polymerase (NEB) for 15 minutes at 72°C and 'A' overhangs were added. The treated PCR products were incubated with TOPO TA vector pCR2.1 and transformed into chemically competent TOP10F' cells as described above. Transformed bacteria were plated on LB agar plates supplemented with ampicillin and then incubated overnight at 37°C. Colonies were grown in liquid culture (LB/ampicillin) and plasmid DNA was prepared (spin miniprep kit). The presence of the insert was confirmed by digestion with HindIII and AfeI and agarose gel electrophoresis. Sequencing was performed on miniprep DNA from many colonies using T7 and M13rev primers. A DNA insert from one such colony has the expected FG27.18 heavy chain sequence; , Qiagen). Maxiprep DNA inserts were confirmed by sequencing.

Light chain cloning of pDCOrig-hIgG1 dual expression vector: FG27.18 light chain was digested from TOPO vector pCR2.1 by sequential digestion with BsiWI and BamHI and the 400 bp insert DNA agarose gel was analyzed using the QIAquick gel extraction kit. (Qiagen) according to the manufacturer's recommendations. This insert was ligated into the previously prepared pDCOrig-hIgG1 vector (see above) and transformed into chemically competent TOP10F' cells. Transformations were plated on LB agar plates supplemented with 35 μg/ml Zeocin and then incubated overnight at 37°C.

Colonies were grown in liquid culture (LB supplemented with 35 μg/ml Zeocin) and plasmid DNA was prepared (spin miniprep kit, Qiagen). Sequencing was performed on miniprep DNA from all colonies using the P6 sequencing primer that maps to the human kappa constant region. DNA inserts from colonies had the expected FG27.18 light chain sequence correctly inserted into pDCOrig-hIgG1; 300 ml bacterial LB/zeocin cultures were grown overnight and plasmid DNA was prepared by maxiprep (plasmid maxi kit, Qiagen).

Cloning pDCOrig-hIgG1 dual expression vector heavy chain: FG27.18 heavy chain insert was digested from TOPO vector pCR2.1 by digestion with HindIII and AfeI. A vector (pDCOrig-hIgG1-27.18k) containing the FG27.18 kappa light chain (prepared above) was also digested with HindIII and AfeI. The vector DNA was then treated with phosphatase according to the manufacturer's recommendations (Antarctic Phosphatase, NEB). After agarose gel electrophoresis, the 6.5 kb pDCOrig-hIgG1 vector band and the 400 bp FG27.18H insert band were isolated using the QIAquick gel extraction kit (Qiagen) according to the manufacturer's recommendations. This insert was ligated into the pDCOrig-hIgG1 vector and transformed into chemically competent TOP10F' cells. Transformants were plated on LB agar plates supplemented with 35 μg/ml Zeocin and then incubated overnight at 37°C. Colonies were grown in liquid culture (LB supplemented with 35 μg/ml Zeocin) and plasmid DNA was prepared (spin miniprep kit, Qiagen). The presence of the insert was confirmed by digestion with HindIII and AfeI and agarose gel electrophoresis. Sequencing was performed on miniprep DNA from a number of colonies using P3rev sequencing primers that map to the human IgG1 constant region. A DNA insert from one of the colonies had the predicted FG27.18 heavy chain sequence correctly inserted into pDCOrig-hIgG1; , plasmid DNA was prepared by maxiprep (plasmid maxi kit, Qiagen). Sequencing was used to confirm the positions of both heavy and light chains.

Expression, Purification and Characterization of Chimeric Antibody Constructs: Methods for expression and purification of chimeric antibodies according to the invention can be accomplished using methods well known in the art. Briefly, antibodies are either transiently or subsequently harvested from supernatants from stable transfected cells using standard protocols, such as Sambrook et al. , 2001 (Sambrook and Russell 2001) by protein A or protein G affinity chromatography.

QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene)
Prepare the reaction as described below:
5 μl 10× reaction buffer 0.12 μl (25 ng) RHA or RKA template 1.3 μl (125 ng) oligonucleotide mutagenic primer For
1.3 μl (125 ng) oligonucleotide mutagenic primer Rev 1 μl dNTPmix
1.5 μl QuikSolution reagent ddH ₂ O (to a final volume of 50 μl)
1 μl of QuikChange Lightning Enzyme

1. Cycle each reaction using the cycling parameters outlined in the table below.

2. Add 2 μl of Dpn I restriction enzyme.
3. Each reaction is mixed gently and thoroughly, briefly microcentrifuged, and immediately incubated at 37° C. for 5 minutes to digest the parental dsDNA.
4. 2 μl of DpnI-treated DNA from each reaction are transformed into separate 45 μl (+2 μl of β-ME) aliquots of XL10-Gold ultracompetent cells (see TOP10™ E. coli Transformation).
5. Colonies are screened using the Phusion method, miniprep, and sequencing to confirm the correct mutation.

Q5® Site-Directed Mutagenesis Kit (NEB Protocol):
1. Assemble the following reagents in a thin-walled PCR tube.

cycle

Kinase, ligase, and DpnI (KLD) treatment2. Add the following reagents and incubate for 5 minutes at RT: 1 μl PCR product 5 μl 2X KLD reaction buffer 1 μl 10X KLD Enzyme Mix
3 μl of nuclease-free water3. 5 μl of the KDL mixture from each reaction was added to separate 50 μl of NEB 5-α Competent E.I. Transform into E. coli. Carefully tap the tube 4-5 times to mix. Do not vortex.
4. Place the mixture on ice for 30 minutes.
5. Heat shock at 42°C for 30 seconds. Keep on ice for 5 minutes.
6. Pipette 950 μl of RT SOC into the mixture.
7. Incubate for 60 minutes at 37° C. with shaking (250 rpm).
8. Mix the cells thoroughly by tapping and inverting the tube, then plate 50 μl on a kanamycin plate and incubate at 37° C. overnight.
9. Colonies are screened using the GoTaq Green method, miniprep, and sequence to confirm the correct mutation.

IgG quantification:
Each well of a 96-well immunoplate is coated with a 100 μl aliquot of 0.4 μg/ml goat anti-human IgG antibody, diluted in PBS and incubated overnight at 4°C. (Plates can be stored for one month at this stage). Similarly, another blank plate is coated with BSA/PBS blocking solution. Excess coating solution is removed and the plate is washed 3 times with 200 μl/well wash buffer. In a blank plate, dispense 120 μl of SEC buffer to all wells, except wells in column 2, row BG. A solution of 1 μg/ml human IgG1/κ antibody in SEC buffer is prepared and served as a standard. Pipette 240 μl/well into column 2 row B and C wells. Media from transfected cells are centrifuged (250 g, 5 min) and the supernatant saved. 240 μl of supernatant from the “no DNA” control (where cos cells were transfected in the absence of DNA) are pipetted into the column 2 row D wells. Pipette 240 μl/well of experimental supernatant into column 2 rows E, F, and G wells. Mix the 240 μl aliquots of the wells in column 2, rows BG and transfer 120 μl to the next well in column 3. Continue to row 11 with a series of 2-fold dilutions of standards, controls, and experimental samples. Transfer 100 μl from each well of the anti-IgG coated plate to the corresponding well. Incubate for 1 hour at 37°C. Wash all wells three times with wash buffer (200 μl). Goat anti-human kappa light chain peroxidase conjugate is diluted 1:5000 in SEC buffer and 100 μl is added to each well. Repeat incubation and washing steps (step 9). Add 150 μl of K-BLUE substrate to each well and incubate for 10 minutes at RT in the dark. The reaction is stopped by adding 50 μl of RED STOP solution to each well. Read the optical density at 655 nm.

Lewis ^y binding ELISA
Each well of a 94-well MaxiSorp plate (Nunc) is coated with 100 ng/well of Lewis Y HSA peptide in PBS and incubated overnight at 4°C. Wash 3 times with PBS-T (0.1% Tween 20). Untreated plates are blocked with 250 μl PBS/0.2% BSA/0.05% Tween 20 per well and incubated for 1 hour at RT. Wash 3 times with PBS-T. 240 μl of antibody (diluted in PBS/0.2% BSA/0.05% Tween 20 if necessary) was added to wells in row 1; 120 μl of buffer (PBS/0.2% BSA/0.05% of Tween 20) is added to the other wells. Transfer 120 μl from column 1 to the next well in column 2. Continue to row 12 with a series of 2-fold dilutions of the experimental sample. Transfer 100 μl per well from the dilution plate to the experimental plate. Incubate for 1 hour at RT. Wash wells 3 times with PBS-T. Goat anti-human Fc peroxidase conjugate is diluted 10000-fold in PBS/0.2% BSA/0.05% Tween 20 (or anti-mouse diluted 10000-fold) and 100 μl is added to each well. Incubate for 1 hour at RT and repeat the wash step. Add 150 μl of substrate (K-Blue) per well and incubate for 150 min at RT. The reaction is stopped by adding 50 μl of RED STOP solution to each well. Read the optical density at 650 nm.

Thermal Stability Fully humanized antibodies and chimeric controls are diluted in TBS/0.2% Tween to 1 μg/ml and aliquoted into PCR tubes in volumes appropriate for the EC80 concentration. Bring the volume up to 100 μl with the same buffer. Each tube is separately heated between 30°C and 85°C for 10 minutes with 5°C intervals and cooled to 4°C. The 1 μg/ml stock is frozen for 1 hour and then diluted to an EC80 concentration. Binding assays against Lewis Y HSA peptides are performed using 100 μl of each antibody per well in a 96-well plate (assayed at each temperature in duplicate).

Comparison of Thermal Shifts Prepare samples directly in white PCR plates of 96-well plates in a final volume of 25 μl (final concentration of purified antibody of 1 and 2 μM). Make a 1:100 stock in Sypro Orange-PBS buffer and then add 1:10 to the final sample (eg 2.5 μl to 25 μl). Load qPCR instrument using MxPro software, SYBR Green method, (filter=FRROX, no reference dye). Setting the thermal profile - 71 cycles of 1°C increments. Plot the results and determine the Tm.

Purification of humanized mabs Instrument: GE Healthcare AKTAxpress™ Purification System
Software: UNICORN
Column: HiTrap MabSelect SuRe, 1 ml; HiLoad 16/600 Superdex 200pg
Mobile phase: IgG elution buffer, Dulbecco's 1x PBS
Sample Prep: Filtered through 0.22 μm Injection Volume: 200 ml Expi293 conditioned medium (1:1) in DPBS
Flow rate: 0.5 ml/min sample loading; 1.5 ml/min gel filtration;
Elute at 1 ml/min

Aggregation Comparison Instruments: Thermostatted column compartment, Wyatt Technology Dawn Heleos, Agilent 1260 infinity HPLC system with Wyatt Technology Optilab TRex Analytical software: Wyatt Technology Astra v1.1.1.
Column: Acquity UPLC BEH200 SEC, 4.6 x 150 mm, 1.7 μm
Mobile phase: Dulbecco's 1×PBS with 0.05% sodium azide
Sample Prep: Filtered through PES 0.22 μm Sample Concentration: Various Injection Volumes: 20 μl
Analysis temperature: 30°C
Flow rate: 0.4ml/min

CIC (Cross-interaction chromatography)
Cross-interaction chromatography (CIC) analysis was performed to assess propensity for non-specific protein-protein interactions and to provide identification of any solubility issues that could lead to downstream manufacturing problems.

（式中、Ｔｒは、ポリＩｇＧのカラムでのサンプルの保持時間であり、Ｔｍは、モック（対照）カラム上の保持時間である。 Samples were injected by two separate 20 μl injections (0.5 mg/ml); was analyzed on a blank of 1 ml of NHS-activated resin coupled to as a control column. The mobile phase consisted of Dulbecco's PBS (Sigma D8537) containing 0.01% sodium azide (0.1 ml/min) and all experiments were performed at 25°C. Eluted samples were detected by UV absorbance (Agilent 1260 Infinity HPLC system with thermostatted column compartment) and data were analyzed using Wyatt Technology ASTRA software (version 6.1.2.83) and sample peak was determined. These were then used to calculate the retention factor k'.

where Tr is the retention time of the sample on the poly-IgG column and Tm is the retention time on the mock (control) column.

Surface Plasmon Resonance (SPR): The Lewis Y HSA conjugate was coupled to a CM5 chip and FG27.10 and FG27.18 mAbs (concentrations ranging from 0.01 μM to 1 μM) were injected at 30 μl/min. Binding data were fitted to a heterologous ligand model using BIAevaluation software.

Antibody internalization: Cancer cells were plated at a density of 2×10 ³ cells/well and allowed to adhere overnight. Cells were cultured in 96-well plates for 72 hours at 37° C. with the indicated concentrations of mAb and secondary antibody: goat antibody to mouse IgG (F'ab fragment) conjugated with saporin (ATSbio, IT#48). or treated with saporin-conjugated goat antibody to human IgG in combination. Control wells consisted of cells incubated with culture medium alone and secondary conjugate alone. The amount of secondary antibody per well was 75 ng and the concentration of primary antibody varied from 0.01 nM to 30 nM. Saporin internalization was assessed via inhibition of thymidine incorporation (thymidine was added during the last 24 hours of incubation).

ADCC and CDC: Cells (5×10 ³ ) were co-incubated with 100 μl PBMC, 10% autologous serum or medium alone, or with a concentration range of mAbs. Maximal spontaneous release was assessed after incubation of labeled cells with medium alone or 10% Triton X-100, respectively. After 4 hours of incubation, 50 μl of supernatant from each well was transferred to the lumaplate of a 96-well plate. Plates were dried overnight and counted on a Topcount NXT counter (Perkin Elmer, Cambridge, UK). The average percentage lysis of target cells was calculated by the following formula:

PI uptake assay: FG27.10 and FG27.18 were incubated with AGS cells and tested for uptake of the small molecular weight dye propidium iodide (PI, Sigma) at various concentrations. The number of PI-positive cells represents the number of dying/dead cells. Tumor cells (5×10 ⁴ ) were incubated on 96-well round-bottom microtiter plates with 50 μl of primary antibody for 2 hours at RT. 1 μg PI was added and cells were incubated for 30 min at RT. Samples were analyzed on an FC-500 flow cytometer (Beckman Coulter). WinMDI 2.9 software was used to analyze and plot the raw data. For comparison, mAbs SC104 and 791T/36, known to induce membrane damage, were also included as internal controls in the experiment.

Growth Inhibition Assay: Growth inhibition by the constructs was assessed by using the water-soluble tetrazolium salt WST-8 (CCK8 kit, Sigma-Aldrich) to measure cellular hydrogenase activity, which is directly proportional to the number of viable cells. . Briefly, after overnight plating of cancer cells (2000 cells/90 μl/well), constructs were added at different concentrations in a final volume of 10 μl/well and plates were incubated at 37° C. (5% CO2) for 72-96 hours. WST-8 reagent was then added (10 μl/well) and after an additional 3 hours incubation the plates were read at 450 nm (Tecan Infinite F50) and the percentage of inhibition calculated. _EC50 values were determined using nonlinear regression (curve fitting) with GraphPad Prism v 8.0 (GraphPad Inc, La Jolla, Calif.).

In vivo model: This study was conducted under the UK Home Office License. We also followed the NCRI guidelines for the welfare and use of animals in cancer research, the LASA good practice guidelines, and the FELAS working group on pain and stress guidelines. Endotoxin-free (<10 EU/ml) FG27 mAb was supplied in preformulated aliquots for administration and stored at −20° C. until use. Age-matched male MF-1 nude mice were obtained from Harlan Laboratories (Bichester, UK) and each group consisted of n≧8 animals, FG27, control mAb or vehicle control.

Mice were implanted with C170HM2 DLuX cells and monitored by optical imaging to determine suitability for tumor establishment and entry into studies. Mice were given either FG27 or a positive control mAb (505/4, FG88.2, or FG88.7 (1 mg/ml) as appropriate) in 100 μl intravenously (i.v.) twice weekly until termination. Dosed at 0.1 mg or mAb vehicle control PBS i.p. twice weekly until terminated. v. was administered in 100 μl. Bioluminescence imaging was performed on all mice weekly to obtain pre- and post-dose tumor measurements. In this way, each mouse provided a pre-dose control reading with which tumor growth could be compared.

All measurements and readings were transferred from the original dictation/notation to Excel (tab delimited) format for data processing in SPSS v16.0. Data integrity was checked using the explore and describe functions. If erroneous points were identified, they were cross-referenced to the original data and corrected accordingly. The data were screened for outliers and distribution profile; data points outside the 95% confidence limits (outliers) were excluded from analysis, but the datasheet was retained for reference.

Mice were imaged weekly for bioluminescent tumor burden (BLI) over the study period as follows: 60 mg/kg D-luciferin substrate was administered subcutaneously (s.c.), mice were anesthetized and BLI was read. were performed 15 min after substrate administration with open filter block (2D) and sequential emission filters (3D reconstruction for DLIT). Ventral and dorsal imaging was performed: the optimal location for imaging was the uppermost abdomen. BLI was measured over the entire abdomen, one region of interest (ROI) for each mouse containing all lesions present. Each mouse had pre-dose or baseline images taken to allow calculation of the percentage of tumor growth over time; these data were averaged per group. BLI readings were also obtained after termination to identify tumors in PM tissue.

Example 1 - Generation and Initial Characterization of FG27 mAb FG27 was generated by immunization of gastric tumor cells with glycolipids.
Analysis of antibody responses to immunization: Antibody titers were initially monitored by enzyme-linked immunosorbent assay (ELISA). Subsequently, thin layer chromatography (TLC) analysis using ST16 total lipid extracts and cell membrane lipid extracts, flow cytometry (FACS) using ST16 tumor cells, and ST16 whole cell extracts, total lipid extracts and cell membrane Western blots using lipid extracts were performed. Mice were boosted intravenously (i.v.) with ST16 cell membrane lipid extract prior to fusion in mice deemed to have the best response compared to pre-bleed serum controls.

Binding of FG27 hybridoma supernatants to a panel of tumor cell lines was analyzed by direct immunofluorescence and FACS analysis. Both FG27.10 and FG27.18 bound ST16 compared to the positive control anti-HLA mAb W6/32 (eBioscience, Calif., USA) and the negative control, but not to human umbilical vein endothelial cells (HUVECs). ) or peripheral blood mononuclear cells (PBMC) (Fig. 4). Both FG27.10 and FG27.18 were cloned. FG27.10 was an IgG3κ and FG27.18 was a subclass of IgG1κ. To ensure that both mAbs bound glycolipids, glycolipids were extracted from a range of cell lines, dried on ELISA plates, and then incubated with FG27.10 and FG27.18. Binding was seen with glycolipids derived from C170, ST16, and AGS with both mAbs, but not with cell lines that showed lack of binding to whole cells (MKN-45, OAW28, OVCAR- 3, OVCAR-4, and OAW42; FIG. 5).

Example 2 - Definition of Epitopes Recognized by FG27 mAbs To determine the degree of specificity of FG27.10, antibodies were first tested for Le ^a , Le ^b , Le ^x , and Le ^y coupled to HSA. Binding to antigen was assessed by ELISA. FG27.10 did not recognize Le ^a , Le ^a−x , or Le ^b , but strongly recognized Le ^y (FIG. 6). To further define the fine specificity of the FG27 mAb, these screens were performed by the Consortium for Functional Glycomics on over 600 natural and synthetic glycans. Binding of FG27.10 and FG27.18 mAbs to the glycan array showed that both mAbs specifically bound to Le ^y (FIG. 7). For comparison, binding of 692/29 showed that the mAb bound most strongly to glycans containing Le ^b , Le ^b , as well as tri-Le ^y and its variants. In contrast, BR96 showed binding to Le ^y and a range of Le ^y variants (Le ^y−x , Le ^y−x−x ), and weaker binding to Le ^x .

FG27.10 and FG27.18 were also screened in a panel of tumor cell lines by SDS/PAGE/Western blot for their property of recognizing glycoproteins and glycolipids (Fig. 8). In addition to bound glycolipids, both mAbs also recognized a range of glycoproteins, as evidenced by binding at the dye front.

Since ^FG27 is specific for Ley, its binding to cell lines (Figure 9 ⁾ was compared to mAb ^BR96 , which recognizes Ley and Lex, and mAb ^692/29 , which recognizes ^Ley and Leb. All three mAbs bound to C170 and AGS cells. In contrast, BR96 bound strongly to Colo201, 692/29 bound moderately, and FG27 did not (Fig. 9). This indicated that this cell line does not express Le ^y , but probably Le ^x and Le ^b , which could not be concluded using non-specific mAbs. HT29 cells were unable to bind either mAb, whereas FG27.10 bound best to OVCAR3 and ST16 cells, and 692/29 bound to LoVo cells.

Example 3 - Immunohistochemical Evaluation of FG27 To evaluate the binding of FG27 to normal human tissues, it was screened in a panel of tissue types. In contrast to mAbs that cross-reacted with Le ^y/b (692/29) and Le ^y/x (BR96), FG27 alone was associated with cell proliferation in normal stomach, lung, tonsil, pancreas, and duodenum. Bound to subpopulations (Table 1). Furthermore, in contrast to other Le ^y cross-reactive mAbs, FG27 failed to stain colon, jejunum, breast, kidney, or ileum. Representative IHC images of normal staining of esophagus, stomach, duodenum, ileum, colon, pancreas, lung, and kidney are shown in FIG.

Table 1: Binding of FG27 to normal human tissues assessed by immunohistochemistry (IHC). Microarray (TMA) staining of these tissues was analyzed via the new viewer software 2010, and the intensity of staining of tumor tissue provided a semi-quantitative score. Strong staining is given a score of 3, moderate staining is given a score of 2, weak staining is given a score of 1, and negative score is 0. The results also demonstrate differential staining of specific cell types within these tissues.

To determine the therapeutic value of FG27, it was screened in human gastric, colorectal, ovarian, and breast TMA by IHC. It was found at different intensities in 80% (69/86) of gastric tumor tissue, 100% (55/55) of colorectal tumor tissue, 95% (36/38) of ovarian tumor tissue and 85% of breast tumor tissue. % (39/46) were stained (Table 2).

Table 2. Binding of FG27 mAb to human gastric, colorectal, ovarian, and breast tumor tissues assessed by immunohistochemistry. Microarray staining of these tissues was analyzed via the new viewer software 2010, and the intensity of staining of tumor tissue provided a semi-quantitative score. Strong staining provides a score of 3, moderate staining provides a score of 2, weak staining provides a score of 1, and negative staining provides a score of 0.

Scoring showed that FG27 broadly bound to gastric tumors, where 59% of the tumors stained positively and 18% of the tumors stained strongly. 19% of the tumors were moderately stained and 22% weakly stained. The results indicate that FG27 targets a wide range of gastric tumors and has the potential to bind strongly to 18% of these. Kaplan-Meier analysis revealed no association between FG27 antigen expression and survival (data not shown). However, there was a correlation between the expression of FG27 antigen and the expression of M30 (a marker of apoptosis) (Pearson chi-square, p=0.049). There was also a correlation between FG27 antigen expression and tumor location (Pearson chi-square, p=0.008), where 65% of stomach-specific tumors stained with FG27 and 26% strongly stained. In contrast, only 53% of tumors in the gastro-esophageal junction were stained, but 37% of these were strongly stained. 46% of tumors in the lower third of the esophagus were stained with FG27 and 53% of these were strongly stained.

Due to the cross-reactivity of FG27 with ovarian cancer cell lines, it was screened against TMA of 360 tumor-bearing ovarian cancers by immunohistochemistry. Scoring showed that FG27.18 bound poorly to ovarian tumors, with 85% of the tumors staining negatively. Only 15% of the tumors were positively stained and only 3% were strongly stained. 3% of the tumors were moderately stained and 9% weakly stained.

Kaplan-Meier survival analysis showed that patients whose tumors expressed the FG27 antigen had significantly better (log-rank, p=0.049) survival than those who did not. . There was a correlation between FG27 antigen expression and FIGO stage (Pearson chi-square, p=0.024). There was also a correlation between FG27 antigen expression and ovarian cancer type (Pearson chi-square, p=0.024), with FG27-expressing mucinous and endometrioid tumors More than 1/3 versus less than 14% of serous clear cells and undifferentiated tumors stained with this mAb. Cox multivariate analysis showed that FG27 did not provide additional prognostic value across FIGO stage, residual disease, and response to chemotherapy.

Table 3: Comparison of tumor staining with the Lewis y-specific mab FG27 and other Lewis cross-reactive mabs BR96 (Lewisy/x) and 692/29 (lewisy/b).

The monospecific Lewis y mab, FG27.10, stained 3-39% more tumors than the Lewis y/b 692/29 mab and 6-48% more tumors than the Lewis y/x Br96 mab, and these results were , reflecting the unique specificity of FG27.10. The greatest changes were seen in ovarian and breast tumors.

Unlike FG27, which showed only moderate binding to normal stomach, pancreas, and duodenum in one of two samples, and weak binding to lung and tonsil in one of two samples, BR96 , esophagus, ileum, jejunum, stomach, breast, lung, tonsil, pancreas, and duodenum. BR96 also showed moderate binding to placenta and gallbladder and weak binding to rectum, kidney, and colon. In contrast, 692/29 bound strongly to the jejunum, moderately to the ileum, stomach, and tonsils, and weakly to the gallbladder, thymus, lung, colon, pancreas, and duodenum (Table 1). . This limited normal binding profile of FG27, particularly for gastrointestinal tissue, did not alleviate antibody binding to tumor tissue, with 80% of stomach tumor tissue and 100% of colorectal tumor tissue exhibiting antigen expression. In contrast, 692/29 and BR96 showed good distribution in colorectal tumor binding at 82% and 90%, respectively (Noble, Spendlove et al. 2013), with similar numbers to the latter, 15/ Eighteen samples, or 83%, are also reported in US Pat. No. 5,491,088. 39% and 49% of these tumors stained moderately or strongly compared to 85% with FG27. 2-25 LE was poorly distributed, binding only 52% of colorectal tumors, with 19% staining moderately and not strongly (Noble, Spendlove et al. 2013).

Thus, it is shown that FG27 has a significantly better normal binding profile, especially in gastrointestinal tissues, and the differential binding between each Le ^y mAb represents distinct properties of the present invention. be

Example 4 - In Vivo Antitumor Activity of FG27 in an Established Colorectal Metastatic Xenograft Model Comparison of Therapeutic Efficacy of mAb FG27 in the C170HM2 DLuX Human Liver Metastasis Model: Using the Mouse C170HM2 DLuX Human Liver Metastasis Tumor Model to investigate the anti-tumor activity of the murine FG27 mAb. The C170HM2 DLuX cell line is passaged to infiltrate the liver parenchyma when implanted into the peritoneal cavity and left to metastasize to the liver for 10 days before initiating mAb treatment. A bioluminescent variant of a colon tumor cell line. The proliferation and distribution/location of such labeled cells and tissues can be assessed in suitable optical imaging systems in real-time and post-mortem (PM) non-invasively in resected tissue.

Anti-tumor data: Twice weekly FG27 administration (100 μg, iv) reduced peritoneal cavity and associated tumor growth compared to vehicle control as compared by bioluminescence intensity (Fig. 11a). ). The study was terminated on day 32. Treatment with FG27 resulted in a significant reduction in bioluminescent tumor burden by the last day of study compared to vehicle controls (p=0.014). There was a significant reduction in tumor growth at the injection site with FG27 treatment (p=0.05; FIG. 11b).

In a second in vivo study with an extended treatment period, FG27 showed a significant reduction in the percentage of tumor growth by day 51 compared to the treated control group (p=0.019) (Fig. 12a). Analysis by log-rank Mantel-Cox test showed significantly higher survival in the FG27-treated group (p=0.0251) compared to vehicle-only controls (Fig. 12b).

Example 5 - Chimeric mAbs
The term "chimeric antibody" refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody. is intended to represent any antibody that is Chimeric (or humanized) antibodies of the invention can be prepared based on the sequences of the murine mAbs prepared as described above. When the gene and amino acid sequences of the antibodies were analyzed, FG27.10 (Figure 1) and FG27.18 (Figure 2) were shown to have identical variable regions but different constant regions IgG3 and IgG1, respectively. rice field. This suggested that a single B cell had switched subclasses and then proliferated during the immunization protocol, and that both of these clones fused successfully.

Compared to the closest germline sequence, FG27 contains 7 mutations (all non-silent) in its heavy chain and 3 mutations (all non-silent) in its light chain. Four amino acid differences relative to germline are in the heavy chain CDRs and three are in FR3. There is a D to H amino acid change at position 36 in CDR1. CDR2 has a G to D amino acid change at positions 59 and 63 and an S to N amino acid change at position 64 (Fig. 2a). In the light chain, there are two amino acid differences in the CDR1 germline, S to I at position 28 and S to T at position 32, and a unique one difference, K51N, in FR2.

There are several other mabs that bind to Lewisy but cross-react with other related Lewis antigens. FG27.10/18 is the only antibody with monospecificity only for Lewis y. This is reflected in the similar but distinct sequences of the FG27 variable heavy and light chain regions (Fig. 13a,b). FG27 has 16-68% differences in amino acid sequence CDRs in the heavy chain, 4.3-16% differences in amino acid differences in light chain CDR sequences, heavy It shows 8.6-49.5% amino acid sequence differences in the variable regions of the chains and 2-24% differences in the amino acid sequences of the light chain variable regions (Table 4). Most of the changes are in CDRH2, with 3-5 of the 8 amino acids varying between different mabs.

Table 4. Amino acid sequence differences in the variable heavy and light chain regions of FG27 from the anti-lewis y cross-reactive mab.

The heavy and light chain variable regions of FG27.18 were cloned into human IgG1 and IgG2 expression vectors. This was transfected into CHO-S cells and human antibodies were protein G purified. The chimeric mAb bound to tumor cell lines expressing Le ^y containing AGS in gastric cell lines (Fig. 14).

Example 6 - Direct Cell Killing Many antiglycan mAbs have been shown to induce direct cell death in antigen-positive cell lines without the need for effector cells or complement. This may enhance their efficacy in vivo, perhaps because tumors may develop mechanisms to evade immune-mediated cell death. This property is primarily associated with glycan-binding mAbs of the mIgG3 isotype, thus the property is lost after chimerization or humanization.

To determine whether FG27.10 and FG27.18 have properties that cause direct cell death, ST16 cells were incubated with FG27.10, FG27.18, and anti-sialyl Le ^a mAb SC104 for 2 h at RT. Cell death was then measured by uptake of propidium iodide, a DNA intercalating agent that is only taken up by dead cells (Fig. 15a). Interestingly, despite having the same variable region, only FG27.10 was able to induce direct cell death and PI uptake was not observed with FG27.18. Figure 15b shows direct killing of a range of antigen-positive (AGS, Colo201, C170, C170HM, MCF-7, OVCAR4) cell lines, where the two mAbs show variable amounts of killing. ing. None of the mAbs showed killing of antigen-negative cell lines (LoVo, MKN45, 791T, SKOV3).

Figure 15c shows PI uptake in the colorectal cell line C170 by a range of mAbs. FG27.10 and positive control antibody SC101 showed strong titratable killing. FG27.18 showed weak, non-titratable killing. The chimeric human IgG1 showed moderate titratable killing and the IgG2 variant did not.

Example 6 - Internalization FG27.10, FG27.18, and CH27 were all evaluated for internalization using various tumor cell lines (Figure 16). FG27.18 dose-stimulates in most cell lines tested, with decreases in cell viability ranging from 60% (C170) to 75% (MCF7) and 85% (AGS) at the highest concentrations tested. showed dependent internalization. FG27.10 showed the best internalization with AGS (up to 50% reduction in viability) and MCF7 (up to 70% reduction in viability). Internalization in the H322 cell line was more variable with both mouse mAbs. CH27 mAb was efficiently internalized by MCF7, where up to 75% reduction in cell viability was observed, and approximately 40% reduction in cell viability was observed for H322.

Example 7 In Vitro Antitumor Activity of FG27 ADCC and CDC: The properties of murine and chimeric FG27 mAbs to induce tumor cell death via ADCC were screened. Human PBMC were used as a source of effector cells and a panel of tumor cells served as target cells. First, ADCC analysis was performed on the supernatant of FG27.10 IgG3 (Fig. 17a). The degree of ADCC seen corresponds to the expression of the Lewis Y antigen and thus is seen with ST16 and OVCAR3, where only low levels were observed with C170 and only background with LoVo, HT29, or Colo201. (Fig. 17a). The extent of ADCC MCF7 and OVCAR3 target cell killing by murine FG27.10 (mIgG3), FG27.18 (mIgG1), CH27 IgG1, and IgG2 was measured and compared after 18 h incubation at 37° C.; CH27 IgG1. provided the highest level of killing of cell lines MCF7 and OVCAR3 (Fig. 17b).

CDC is known to be an important mechanism involved in the elimination of tumor cells in vivo. ST16 killed by CDC induced by mAbs FG27.10, FG27.18, CH27 IgG1, and CH27 IgG2 in the presence or absence of human serum as a source of complement for 18 hours at 37°C. Cell properties were assayed. As shown in FIG. 17c, among the FG27 mAbs, FG27.10 (mIgG3) provided the highest level of killing at 3 μg/ml, whereas no significant levels were seen with CH27 IgG2, IgG2 This was expected given the nature of the antibody. Similarly, when using AGS cells, FG27.10 provided the highest level of killing, followed by CH27 IgG1. Again, no killing was seen with CH27 IgG2 (Fig. 17d).

Taken together, FG27 mAb potently induced ADCC and significant CDC using human PBMC as effector cells with human serum as complement source.

実施例９－ＦＧ２７抗体のヒト化バージョンの作製 ヒトＶＨおよびＶＫ ｃＤＮＡのデータベース Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｉｍｍｕｎｏｇｅｎｅｔｉｃｓ Ｄａｔａｂａｓｅ ２００９ （Ｌｅｆｒａｎｃ ３０７－１０）およびｔｈｅ Ｋａｂａｔ Ｄａｔａｂａｓｅ Ｒｅｌｅａｓｅ ５ ｏｆ Ｓｅｑｕｅｎｃｅｓ ｏｆ Ｐｒｏｔｅｉｎｓ ｏｆ Ｉｍｍｕｎｏｌｏｇｉｃａｌ ｉｎｔｅｒｅｓｔ（ｌａｓｔ ｕｐｄａｔｅ １７－Ｎｏｖ－１９９９ ) (Kabat et al. 1-3242) were used to compile a database of Kabat-aligned human immunoglobulin sequences. Our database contains 10,906 VH sequences and 2,912 VK sequences.

Selection of FG27 Human Frameworks Humanization requires identification of suitable human V regions. The sequence analysis program Gibbs was used to search the human VH and VK databases with the FG27 VH and VK protein sequences using various selection criteria. Using the program Discovery Studio (Accelrys), FW residues (Kabat definition) within 4 Å of the CDR residues of the structure of the murine FG27 antibody were identified and named "4 Å Proximity Residue". did. The human VH sequences with the highest identity to the FG27 VH at the 4 Å proximal residues are identical to these envelope residue and VCI lists, as well as the mouse equivalent positions of the FW, VCI, or 4 Å proximal residues. are shown in FIG. 19 along with the number of residues of . FIG. 18 shows the alignment and residue identities of all VH sequences of FIG. The number of apparent somatic mutations in each FW identified by comparison with the most identical human germline V genes is shown in FIG.

Humanized, murine and incomplete sequences were excluded from this analysis. Sequence AJ579110 was selected as a human donor candidate due to the high sequence identity and similarity of this sequence. It has no somatic mutations from its germline ABO19439 or HM855436.

Design of FG27 RHA and FG27 RHB Having identified suitable human frameworks, synthetic protein and DNA sequences can be designed. The initial design of a humanized version of FG27 is the grafting of CDR1, 2 and 3 from the FG27 VH onto the FW of the acceptor AJ579110. One VCI+4 Å proximal residue at Kabat position 24 is not conserved in FG27 RHA and is backmutated to the mouse equivalent residue to generate a humanized version of FG27 RHB. Figure 21 compares the murine and humanized versions of the FG27 VH protein sequence.

Selection of FG27 RKA Framework To humanize the light chain, the human kappa chain was identified in a similar process as the heavy chain. Figures 22, 23 show the 4A-proximal residues and the number of residues in the FW or 4A-proximal residues that are identical to the mouse equivalent positions, alignment of the VK sequences, and comparison to the human germline. Initial analysis found several potential donor candidates, all of which proved to be poorly expressed human VK4. Expanding the analysis to include CDR1 with one less residue resulted in a single candidate, X72449, which exhibited a higher degree of sequence homology to the murine antibody than the VK4 candidate. This candidate had a single somatic mutation from the human VK2 germline but no somatic mutation from the human VK2D germline (Figure 24).

Design of FG27 RKA and FG27 RKB The framework from X72449 was used to design the DNA and protein of the humanized constructs. CDR1, 2, and 3 from FG27 VK have been shown to be grafted onto the acceptor FW of X72449 to produce an early version of humanized FG27. In FG27 RKA there is a single mismatched 4 Å proximal residue 5, whereas in variant FG27 RKB this residue was backmutated to the equivalent murine residue (Fig. 25).

Generation and Properties of Humanized Versions of FG27 Generation of FG27 Humanized Antibodies The genes for FG27 RHA and FG27 RKC were synthesized by Genewiz and codon optimized with respect to the human sequence (Fig. 3ab). The heavy and light chains of the native human framework sequences M65092 and X72449, respectively, and the native mouse CDR sequences were assembled in silico and designated FG27 RHA-FG27 RHB and FG27 RKA-FG27 RKD. Using Genewiz's proprietary software algorithms, sequences were optimized by silent mutagenesis to use codons that are preferentially utilized and synthesized by human cells. The RHA/B and RKA/B constructs were amplified by PCR using primers specific for the expression vector + insert (as previously described for the chimeric version) and ligated into pHuG1 and pHuK in a ligase independent cloning reaction. was used to transform TOP10 bacteria. RKA and RHA were then modified by PCR mutagenesis to obtain all human variants represented in FIGS.

Clones were sequenced and expression plasmid DNA was prepared using the QIAGEN Plasmid Miniprep Kit or Qiagen Plasmid Maxiprep kit. Expression construct sequences (RHA, RHB, RKA and RKB) are shown in FIGS. Expression plasmid preparations encoding (humanized or chimeric) VH and VK were used to transfect Expi293 cells and cultured in serum-free medium for 10 days, after which conditioned medium containing secreted antibody was harvested.

Antibody Expression Concentrations of IgG1κ antibodies in the conditioned medium of Expi293 cells were measured by ELISA and are shown in Table 5. Most antibodies were produced at sufficient levels, except those highlighted in red.

Table 5. Levels of IgG in conditioned medium of transfected Expi293 cells

Binding of Lewis Y-HSA by FG27 Antibody Binding activity to the Lewis Y-HSA antigen was measured by binding ELISA. Initial experiments showed essentially no difference between binding potencies of murine or chimeric antibodies. This result gave us some confidence in the success of the humanization process.

The data shown in Figure 26 demonstrate the binding potency of the first version of humanized FG27. Although all versions bound to Lewis Y-HSA, antibody versions consisting of RHA/RKB, RHA/RKC, RHA/RKD, and RHB/RKC were selected as possible lead candidates.

Thermal Stability of Candidate Humanized Antibodies Up to High Temperatures The purpose of this experiment is to compare the thermal stability of humanized antibodies. When subjected to elevated temperatures varying from 30-85° C. for 10 minutes, they are cooled to 4° C. and used in the ELISA assay at the EC80 concentration of each candidate. All antibodies tested appeared equally stable (Figure 27), with all becoming inactive above 72°C.

Determination of Candidate Humanized Antibody Tm To determine the melting temperature of the chimeric and humanized lead antibodies, all antibodies RHA/RKB, RHA/RKC, RHA/RKD, and RHB/RKC were subjected to two-step affinity chromatography. Purified by graphics and gel filtration system and tested by thermal shift assay. Samples were incubated with a fluorescent dye (Sypro Orange) for 71 cycles in a qPCR thermal cycler with 1°C increments per cycle. The Tms of the chimeric and four humanized antibodies are shown in FIG. All of the candidate and chimeric antibodies have the same Tm (72°C).

Affinity of Humanized Candidate Antibodies Antibody affinity determinations using SPR analysis were performed using a Biacore T3000. Lewis Y-HSA was covalently immobilized to a CM5 chip (Cat: BR-1005-30) according to the manufacturer's instructions. Chimeric FG27, RHA/RKB, RHA/RKC, RHA/RKD, and RHB/RKC antibodies were diluted in buffer at concentrations ranging from 200 nM to 0.274 nM (3-fold serial dilutions), then 50 μl /min flow rate. The binding time was set at 120 seconds and the dissociation time was set at 600 seconds.

Kinetics of association/dissociation were analyzed by best fitting heterologous ligand models using the BIAcore 3000 evaluation software package. An overlay of the 200 nM binding curve is shown in the sensogram (Figure 29). RHA/RKB, RHA/RKC and RHB/RKC show similar affinities to chimeric FG27 (RHA/RKB>RHA/RKC>RHB/RKC>>RHA/RKD). These results suggest that humanization successfully retains binding activity within desirable parameters, with RHA/RKB or RHA/RKC having the highest affinity.

Aggregation of Candidate Humanized Antibodies Samples were injected at 0.4 ml/min onto the size exclusion column of the HPLC system and analyzed by multi-angle light scattering to determine absolute molar mass and confirm aggregation. All variants show no evidence of aggregation with average molar molecular weights in the range of 140.19 kDa, the expected range for IgG monomers in the context of this analysis. All samples are monodisperse (Mw/Mn<1.05). The mass recovery (mass calculated/mass injected) represents good protein recovery and the sample does not appear to stick to the column or contain insoluble aggregates retained by the guard column. 100%. Collective data suggest that there is no aggregation concern for any of the anti-FG27 antibody samples analyzed.

Non-specific protein-protein interactions (CIC)
Cross-Interaction Chromatography (CIC) using bulk-purified human polyclonal IgG is a technique for monitoring non-specific protein-protein interactions and is used to distinguish between soluble and insoluble antibodies. can be used. A high retention index (k') indicates a propensity for self-interaction and low solubility. The RHA/RKB, RHA/RKC, RHB/RKC antibodies exhibited a retention index of less than 0.044, indicating low propensity for non-specific interactions and good solubility (Figure 30).

Antibodies have been engineered and expressed as fully humanized antibodies without loss of Lewis Y HSA binding potency (Figures 26 and 29). Experiments with chimeric antibodies consisting of murine variable regions on human constant regions showed the same efficacy of the Lewis Y HSA binding ELISA as the murine antibody. This result provides some confidence in the positive outcome of the humanization process.

Initial experiments demonstrate that fully humanized heavy and light chains, i.e., without framework mutations to introduce mouse 4A proximal residues, do not bind to the Lewis Y HSA peptide. , as well as a version of the chimeric positive control antibody but containing a single backmutation binds equivalently to the chimeric positive control. Assays measuring antibody solubility, thermal stability, and aggregation suggest that all humanized candidates possess acceptable biophysical characteristics.

In our view, the combination of excellent binding, expression, thermal stability, affinity, and cell binding make the RHA heavy chain version in combination with the RKC light chain version the best candidate for further development. ing.

Example 10 - FG27 Binding Studies A humanized mAb (humanized 27) and a chimeric mAb (CH27) showed similar cell binding patterns at titration in cancer cell lines (Figure 31). Equilibrium affinity constants are overall in the same order of magnitude but slightly higher compared to the parental murine antibody, and the maximal binding capacities of humanized 27 and CH27 are greater compared to those of the murine mAbs. This result suggests a successful outcome for the humanization process.

Example 11 - Direct killing of humanized mabs To enhance direct cell killing of the human IgG1 27 chimera (27hIgG1), we added selected mIgG3 constant region residues to the hIgG1 Fc domain. An improved 'i27G1' Lewis ^Y glycan-binding mAb with improved direct cell-killing capacity in vitro was generated by transfer. Figure 33 shows the amino acid and nucleic acid sequences of each light and heavy chain of the "i27G1" antibody. This antibody forms another aspect of the invention.

FIG. 32 shows improved direct cell killing, functional affinity, and effector function of i27G1. The mIgG3 27 antibody, but not the chimeric 27hIgG1, has direct cell-killing ability in Lewis ^y -expressing AGS cell lines (i) and MCF7 cell lines (ii). Our i27G1, which contains selected mIgG3 constant region residues, showed a significantly improved direct response compared to 27hIgG1, comparable to 27mG3 mAb, in both AGS (i) and MCF7 (ii). exhibit significant cell killing. Furthermore, i27G1 exhibits significantly improved functional Lewis ^y affinity compared to 27hIgG1(iii). Importantly, the effector functions, ADCC, and CDC of i27G1 were all comparable to those of 27hIgG1 at MCF7 (iv and v). Taken together, these results suggest that the transfer of selected regions from the mIgG3 constant region to the 27hIgG1 backbone yields hybrid mAbs with direct cell-killing potential, increased functional affinity, and potent immune effector functions. represents what has been brought about.

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Claims (21)

An isolated specific binding member capable of specifically binding to Fuca1-2Galb1-4(Fucal-3)GlcNAc(Le ^y ). one or more binding domains selected from binding domains having an amino acid sequence substantially represented as residues 27-38 (CDRH1), 56-65 (CDRH2), or 105-116 (CDRH3) of FIG. 1a or 1b 2. A binding member according to claim 1, comprising a domain. 3. A binding member according to claim 2, comprising the amino acid sequence substantially presented as residues 1-127 (VH) of FIG. 1a or 1b. one or more binding domains selected from binding domains having an amino acid sequence substantially represented as residues 27-38 (CDRL1), 56-65 (CDRL2), or 105-113 (CDRL3) of FIG. A binding member according to any preceding claim comprising. 5. A binding member according to claim 4, comprising the amino acid sequence substantially presented as residues 1-124 (VL) of FIG. 1c. A binding member according to any preceding claim comprising residues 1-127 (VH) of the amino acid sequence of Figure 1a or 1b and residues 1-124 (VL) of the amino acid sequence of Figure 1c. 5. A binding member according to claim 2 or 4, wherein at least one binding domain is carried by a human antibody framework. A binding member according to any preceding claim, further comprising a human constant region. 4. A binding member according to any preceding claim, wherein said binding member is an antibody or antibody fragment. 10. A binding member according to claim 8 or 9, comprising a heavy chain amino acid sequence substantially as presented in Figure 2a and a light chain amino acid sequence substantially as presented in Figure 2b. 5. A binding member according to claim 1, 2 or 4, comprising a heavy chain amino acid sequence substantially as presented in Figure 3a and a light chain amino acid sequence substantially as presented in Figure 3b. An isolated binding member capable of specifically binding Fuca1-2Galb1-4(Fucal-3)GlcNAc(Le ^y ) that competes with the isolated specific binding member of any one of claims 2-11. a specific binding member. A binding member according to any preceding claim which is attached to or otherwise associated with a chemotherapeutic or cytotoxic agent. A pharmaceutical composition comprising a binding member according to any preceding claim and a pharmaceutically acceptable excipient, diluent, carrier, buffer or stabilizer. A binding member according to any one of claims 1 to 12 for use in medicine. A binding member according to any one of claims 1 to 12 for use in a method of treating or preventing tumors. A product comprising a specific binding member according to any one of claims 1-12 and an active agent as a combination formulation for simultaneous, separate or sequential use in the treatment of tumors. 17. A binding member for use according to claim 14 or 15 or a product according to claim 16, wherein said tumor is a colorectal, gastric, pancreatic, lung, ovarian or breast tumor. A nucleic acid encoding a binding member according to any one of claims 1-12. A binding member comprising a heavy chain having an amino acid sequence substantially as presented in Figure 33 and/or a light chain having an amino acid sequence substantially as presented in Figure 33. 21. A binding member according to claim 20, comprising a heavy chain encoded by a nucleic acid sequence substantially as presented in Figure 33 and/or a light chain encoded by a nucleic acid sequence substantially as presented in Figure 33.

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